U.S. patent application number 16/343747 was filed with the patent office on 2019-08-15 for multi-configurable sensing array and methods of using same.
This patent application is currently assigned to EnLiSense, LLC. The applicant listed for this patent is Board of Regents, The University of Texas System, EnLiSense, LLC. Invention is credited to Sriram Muthukumar, Shalini Prasad.
Application Number | 20190250153 16/343747 |
Document ID | / |
Family ID | 62019012 |
Filed Date | 2019-08-15 |
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United States Patent
Application |
20190250153 |
Kind Code |
A1 |
Muthukumar; Sriram ; et
al. |
August 15, 2019 |
MULTI-CONFIGURABLE SENSING ARRAY AND METHODS OF USING SAME
Abstract
Disclosed herein are devices, apparatus, systems, methods and
kits for performing immunoassay tests on a sample. The A sensing
apparatus is provided for detecting a plurality of different target
analytes in a sample. The apparatus may comprise an array of
sensing devices provided on a substrate, each sensing device in the
array comprising a working electrode having (1) semiconducting
nanostructures disposed thereon and (2) a capture reagent coupled
to the semiconducting nanostructures that selectively binds to a
different target analyte in the sample. The apparatus may also
comprise sensing circuitry that (1) simultaneously detects changes
to electron and ion mobility and charge accumulation in the array
of sensing devices when the capture reagents in the array of
sensing devices selectively bind to the plurality of different
target analytes, and (2) determines the presence and concentrations
of the plurality of different target analytes in the sample based
on the detected changes.
Inventors: |
Muthukumar; Sriram; (Allen,
TX) ; Prasad; Shalini; (Allen, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EnLiSense, LLC
Board of Regents, The University of Texas System |
Allen
Austin |
TX
TX |
US
US |
|
|
Assignee: |
EnLiSense, LLC
Allen
TX
Board of Regents, The University of Texas System
Austin
TX
|
Family ID: |
62019012 |
Appl. No.: |
16/343747 |
Filed: |
October 19, 2017 |
PCT Filed: |
October 19, 2017 |
PCT NO: |
PCT/US2017/057478 |
371 Date: |
April 19, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62410598 |
Oct 20, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/68 20130101; G01N
27/3335 20130101; G01N 33/543 20130101; G01N 33/5438 20130101; G01N
33/588 20130101; G01N 27/302 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; C12Q 1/68 20060101 C12Q001/68; G01N 27/30 20060101
G01N027/30; G01N 27/333 20060101 G01N027/333 |
Claims
1.-52. (canceled)
53. An apparatus comprising: an array of a plurality of sensing
devices provided on a substrate, wherein each sensing device in the
array comprises: a working electrode with semiconducting
nanostructures disposed thereon; a capture reagent coupled to the
semiconducting nanostructures to selectively bind to a respective
target analyte in a sample; and a respective counter electrode; and
at least one common reference electrode shared by two or more of
the plurality of sensing devices.
54. The apparatus of claim 53, wherein the sensing circuitry is to
simultaneously detect changes to electron and ion mobility and
charge accumulation in the array of sensing devices when the
capture reagents in the array of sensing devices selectively bind
to corresponding target analytes.
55. The apparatus of claim 53, wherein the apparatus is to detect
any one of a plurality of different analytes present in the sample
using the array of sensing devices and sensing circuitry.
56. The apparatus of claim 55, wherein the array of sensing devices
comprises sensing devices with different capture reagents to
selective bind to different analytes in the plurality of
analytes.
57. The apparatus of claim 55, wherein the apparatus is configured
to detect the plurality of different target analytes in the sample,
wherein the sample has a volume of less than 30 pL.
58. The apparatus of claim 53, further comprising sensing circuitry
to determine both presence and concentration of one or more of the
respective target analytes from the array of sensing devices.
59. The apparatus of claim 53, wherein the common reference
electrode is located between the respective working electrode and
the counter electrode of the two or more sensing devices.
60. The apparatus of claim 59, wherein the two or more sensing
devices comprise a first sensing device and a second sensing
device, the working electrode and the counter electrode of a first
sensing device are located in proximity to each other in a first
region of the substrate, and the working electrode and the counter
electrode of a second sensing device are located in proximity to
each other in a second region of the substrate.
61. The apparatus of claim 60, wherein the common reference
electrode is located in an overlapping region between the first and
second regions.
62. The apparatus of claim 60, wherein the first sensing device
comprises a first capture reagent that selectively binds to a first
target analyte in the sample, and the second sensing device
comprises a second capture reagent that selectively binds to a
second target analyte in the sample.
63. The apparatus of claim 62, wherein the first and second target
analytes are different biomarkers.
64. The apparatus of claim 62, wherein the first and second target
analytes are different isoforms of a same type of biomarker.
65. The apparatus of claim 53, wherein two or more sensing devices
in the array comprise working electrodes having the same type of
semiconducting nanostructures.
66. The apparatus of claim 53, wherein two or more sensing devices
in the array comprise working electrodes having different types of
semiconducting nanostructures.
67. The apparatus of claim 53, wherein the sample comprises at
least one of sweat, blood, serum, or urine of a human subject.
68. A method comprising: providing a sample on a test device
comprising an array of sensing devices provided on a substrate;
sensing, using the array of sensing devices, any one of a plurality
of different target analytes, wherein each sensing device in the
array comprises a working electrode with semiconducting
nanostructures disposed thereon and a capture reagent coupled to
the semiconducting nanostructures to selectively bind to and sense
a respective target analyte in the sample; and determining, from
the array of sensing devices both presence and concentration of one
or more of the respective target analytes from the array of sensing
devices.
69. A system comprising: a module comprising: an array of a
plurality of sensing devices provided on a substrate, wherein the
each sensing device in the array comprises: a working electrode
with semiconducting nanostructures disposed thereon; a capture
reagent coupled to the semiconducting nanostructures to selectively
bind to a respective target analyte in a sample; and a respective
counter electrode; and at least one common reference electrode
shared by two or more of the plurality of sensing devices; and
sensing circuitry to determine both presence and concentration of
one or more of the respective target analytes from the array of
sensing devices.
70. The system of claim 69, wherein the module and sensing
circuitry are present on a particular device.
71. The system of claim 70, wherein the particular device comprises
a wearable device to be worn on a portion of a user's body.
72. The system of claim 69, further comprising a portable health
diagnostics system comprising the sensing circuitry, wherein the
portable health diagnostics system is to inspect the module.
73. A non-transitory computer readable medium storing instructions
that, when executed by one or more processors, causes the one or
more processors to: collect electrical signals from an array of
sensing devices provided on a substrate, each sensing device in the
array comprising a working electrode comprising: semiconducting
nanostructures disposed thereon and a capture reagent coupled to
the semiconducting nanostructures to selectively bind to a
respective target analyte; simultaneously detect changes to
electron and ion mobility and charge accumulation from the
collected electrical signals when the capture reagents in the array
of sensing devices selectively bind to the corresponding target
analytes in the sample; and determine the presence and
concentrations of the plurality of different target analytes in the
sample based on the detected changes.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application 62/410,598 filed on Oct. 20, 2016, the content of which
is incorporated herein in its entirety.
BACKGROUND
[0002] Early detection and reliable diagnosis can play a central
role in making effective therapeutic decisions for treatment of
diseases or managing certain physiological conditions. Detection
may involve identification of disease-specific biomarkers in human
body fluids that indicate irregularities in cellular regulatory
functions, pathological responses, or intervention to therapeutic
drugs.
[0003] Immunoassays can provide rapid and cost-effective mechanisms
for detecting the presence and concentrations of analytes in a
sample. Oftentimes, a single analyte (e.g. biomarker) or molecule
may not be sufficient for unambiguous identification of specific
diseases or for treating complex pathology conditions. In many
cases, it is desirable to simultaneously detect the presence and
concentration of more than one analyte in a sample, for example a
variety of different analytes. More sensitive methods and devices
for performing such tests are needed, that can enable users to
perform quantitative measurements with higher accuracy and wider
dynamic range than currently available biosensing devices.
[0004] Wearable sensors that monitor disease-specific biomarkers
can be used for maintaining stasis in humans and their surrounding
environments. Common modalities for biological/chemical sensing may
utilize affinity-based reactions and binding mechanisms to
transduce optical, electrical, and/or mechanical signals. There is
a need for wearable and non-invasive (or minimally invasive)
sensing technologies that allow users to accurately and rapidly
evaluate their physiological status in a continuous manner.
Ideally, such analysis and quantification may be performed in
real-time in order to provide prompt feedback to the users.
SUMMARY
[0005] The present disclosure addresses at least some of the above
needs. Various embodiments of the present disclosure address the
demand for wearable and non-invasive sensors that are capable of
quantifying multiple different types of target agents (chemical,
biological, etc.) simultaneously and in real-time. The sensing
devices and methods described herein can enable detection of (i) a
wide range of chemical agents and/or (ii) a wide range of
biomarkers (analytes) that provide indicators about a person's
physiological state, for detecting diseases and also for monitoring
the health conditions of the user/wearer. In some embodiments,
microelectrode affinity-based electrical sensing platforms for
point-of-care (POC) detection of disease-specific biomarkers can
provide quantitative, multiplexed, and simultaneous detection of
multiple biomarkers for rapid diagnostic and prognostic analysis on
a single test sample that is introduced onto the sensing platform.
Point-of-care, rapid quantification of protein biomolecules (that
are specific biomarkers of certain diseases) can help in various
aspects of disease diagnosis, monitoring, and analysis. The
multiplexed and simultaneous detection of multiple biomarkers on a
common sensing platform obviates the need to have multiple discrete
immunoassay strips for detecting different biomarkers, and may also
eliminate the need to collect multiple samples for testing.
[0006] In some embodiments, the multi-biomarker sensing devices and
methods described herein can weigh individual biomarkers
differentially on the basis of the end physiological state being
predicted. In some embodiments, highly specific biomarkers can be
detected rapidly at ultralow concentrations from very low fluid
sample volumes from a user. Disease specific protein biomarker
detection can be achieved having (1) ultra-sensitivity in reliable
detection at low concentrations (typically in lower pg/ml), and (2)
specificity in protein detection from complex solutions such as
body fluids.
[0007] According to some aspects of the disclosure, a sensing
apparatus for detecting a plurality of different target analytes in
a sample is provided. The apparatus may comprise an array of
sensing devices provided on a substrate. Each sensing device in the
array may comprise a working electrode having (1) semiconducting
nanostructures disposed thereon and (2) a capture reagent coupled
to the semiconducting nanostructures that selectively binds to a
different target analyte in the sample. The apparatus may also
comprise sensing circuitry that (1) simultaneously detects changes
to electron and ion mobility and charge accumulation in the array
of sensing devices when the capture reagents in the array of
sensing devices selectively bind to the plurality of different
target analytes, and (2) determines the presence and concentrations
of the plurality of different target analytes in the sample based
on the detected changes.
[0008] Also disclosed is a method of detecting a plurality of
different target analytes in a sample. The method may comprise:
providing the sensing apparatus described herein; applying the
sample to the array of sensing devices; and with aid of the sensing
circuitry, simultaneously detecting the changes to the electron and
ion mobility and charge accumulation in the array of sensing
devices by simultaneously measuring (1) impedance changes using a
modified Electrochemical Impedance Spectroscopy (EIS) technique and
(2) capacitance changes using a Mott-Schottky technique; and
determining the presence and concentrations of the plurality of
different target analytes by concurrently analyzing the measured
impedance and capacitance changes.
[0009] In some embodiments, a sensing system may comprise: a test
strip comprising the array of sensing devices, and a point-of-care
(POC) portable health diagnostics reader comprising the
aforementioned sensing circuitry, wherein the diagnostics reader
comprises an opening for receiving the test strip.
[0010] In some embodiments, a wearable device may comprise the
aforementioned sensing apparatus and may be configured to be worn
on a portion of a user's body.
[0011] In another aspect, a non-transitory computer readable medium
storing instructions that, when executed by one or more processors,
causes the one or more processors to perform a computer-implemented
method for detecting a plurality of different target analytes in a
sample is provided. The method may comprise: collecting electrical
signals from an array of sensing devices provided on a substrate,
each sensing device in the array comprising a working electrode
having (1) semiconducting nanostructures disposed thereon and (2) a
capture reagent coupled to the semiconducting nanostructures that
selectively binds to a different target analyte; simultaneously
detecting changes to electron and ion mobility and charge
accumulation from the collected electrical signals when the capture
reagents in the array of sensing devices selectively bind to the
different target analytes in the sample; and determining the
presence and concentrations of the plurality of different target
analytes in the sample based on the detected changes.
[0012] Further aspects of the disclosure are directed to a modular
sensing kit for detecting a plurality of different target analytes
in a sample. The kit may comprise: a base module comprising at
least one reference electrode and at least one counter electrode
disposed on a substrate; and a plurality of discrete sensors
configured to be interchangeably and releasably coupled to the base
module, each of the plurality of discrete sensors comprising a
working electrode having (1) semiconducting nanostructures disposed
thereon and (2) a capture reagent coupled to the semiconducting
nanostructures that selectively binds to a different target analyte
in the sample.
[0013] In some embodiments, the working electrodes of the plurality
of discrete sensors may have the same type or different types of
semiconducting nanostructures. Each of the plurality of discrete
sensors can be configured to be mechanically and electrically
coupled to the base module. Each of the plurality of discrete
sensors is usable for determining a presence and concentration of a
different target analyte in the sample. In some cases, the base
module may comprise at least one receiving portion on the substrate
for coupling to a discrete sensor. Additionally, the base module
may comprise a plurality of receiving portions on the substrate for
coupling to a plurality of discrete sensors.
[0014] A module sensing device may comprise the base module, and a
discrete sensor that is selected from the plurality of discrete
sensors and coupled to the base module. A modular sensing apparatus
may comprise the base module, and two or more discrete sensors that
are selected from the plurality of discrete sensors and coupled to
the base module, to thereby provide an array of sensing devices. In
some embodiments, at least two sensing devices from the array may
utilize a common reference electrode. The common reference
electrode may be located between the working electrodes of the at
least two sensing devices. The modular sensing apparatus may
further comprise sensing circuitry that (1) simultaneously detects
changes to electron and ion mobility and charge accumulation in the
array of sensing devices when the capture reagents in the array of
sensing devices selectively bind to the plurality of different
target analytes, and (2) determines the presence and concentrations
of the plurality of different target analytes in the sample based
on the detected changes.
[0015] A method of detecting a target analyte in a sample may
comprise: providing the modular sensing kit; forming the modular
sensing device by coupling the selected discrete sensor to the base
module; applying the sample to the modular sensing device; and with
aid of sensing circuitry, detecting changes to electron and ion
mobility and charge accumulation in the modular sensing device by
measuring (1) impedance changes using a modified Electrochemical
Impedance Spectroscopy (EIS) technique and (2) capacitance changes
using a Mott-Schottky technique; and determining a presence and
concentration of a target analyte by analyzing the measured
impedance and capacitance changes.
[0016] A method of detecting a plurality of target analytes in a
sample may comprise: providing the modular sensing kit; forming the
modular sensing apparatus by coupling the selected two or more
discrete sensors to the base module; applying the sample to the
array of sensing devices; and with aid of the sensing circuitry,
simultaneously detecting the changes to the electron and ion
mobility and charge accumulation in the array of sensing devices by
simultaneously measuring (1) impedance changes using a modified
Electrochemical Impedance Spectroscopy (EIS) technique and (2)
capacitance changes using a Mott-Schottky technique; and
determining the presence and concentrations of the plurality of
different target analytes by concurrently analyzing the measured
impedance and capacitance changes.
[0017] In some embodiments, a sensing system may comprise: a test
strip comprising the modular sensing apparatus; and a point-of-care
(POC) portable health diagnostics reader comprising the sensing
circuitry, wherein the diagnostics reader comprises an opening for
receiving the test strip. In some embodiments, a wearable device
may comprise the modular sensing apparatus and may be configured to
be worn on a portion of a user's body.
[0018] According to some aspects of the disclosure, a sensing
device for detecting one or more target analytes in a fluid sample
is provided. The device may comprise a substrate comprising two or
more electrodes, a plurality of semiconducting nanostructures
disposed on at least one of the electrodes, and a plurality of
capture reagents attached to the plurality of semiconducting
nanostructures. The plurality of capture reagents are configured to
selectively bind to the one or more target analytes in the fluid
sample, thereby effecting changes to electron and ion mobility and
charge accumulation in different regions of the semiconducting
nanostructures and the fluid sample. The changes to the electron
and ion mobility and charge accumulation can be detected with aid
of sensing circuitry, and used to determine a presence and
concentration of the one or more target analytes in the fluid
sample. The changes may comprise simultaneous modulation to the ion
mobility in one or more regions adjacent or proximal to the
semiconducting nanostructures.
[0019] In some embodiments, the changes to the electron and ion
mobility and charge accumulation can be transduced into electrical
impedance and capacitance signals. The signals may be indicative of
interfacial charge modulation comprising of the changes to the
electron and ion mobility. The signals may be indicative of
capacitance changes to a space-charge region formed in the
semiconducting nanostructures upon binding of the one or more
target analytes to the capture reagents. The sensing circuitry can
be configured to implement a plurality of electrochemical detection
techniques for detecting the capacitance changes and impedance
changes. The plurality of electrochemical detection techniques may
include (1) a modified Electrochemical Impedance Spectroscopy (EIS)
technique for measuring the impedance changes and (2) Mott-Schottky
technique for measuring the capacitance changes. The sensing device
is capable of simultaneous and multiplexed detection of a plurality
of target analytes present in the fluid sample using the plurality
of electrochemical detection techniques. In some instances, the
sensing device comprises the sensing circuitry, and the sensing
circuitry can be configured to perform the simultaneous and
multiplexed detection by analyzing the electrical impedance and
capacitance signals to determine the presence and concentration of
each of the plurality of target analytes. The sensing circuitry can
be configured to perform the simultaneous and multiplexed detection
substantially in real-time upon binding of the plurality of target
analytes to the capture reagents on the semiconducting
nanostructures.
[0020] In some embodiments, the sensing circuitry can be configured
to analyze the electrical impedance and capacitance signals by
concurrently analyzing a set of Nyquist plots obtained via the
modified EIS technique and a set of Mott-Schottky plots obtained
via the Mott-Schottky technique. The modified EIS technique may
comprise (1) sectioning an interfacial charge layer into a
plurality of spatial dielectric z-planes along a direction
orthogonal to the interface between the fluid sample and the
semiconducting nanostructures, and (2) probing each of the
plurality of z-planes with a specific frequency selected from a
range of frequencies. Specific binding of different target analytes
to the capture reagents occurs at known spatial heights within the
interfacial charge layer, and the sensing circuitry can be
configured to determine the presence and concentration of each of
the different target analytes by measuring the capacitance and
impedance changes at specific frequencies corresponding to their
respective z-planes at the known spatial heights within the
interfacial charge layer. The modified EIS technique is capable of
distinguishing the electrical impedance signals from background
noise at low concentrations of the target analytes in the fluid
sample.
[0021] In some embodiments, the sensing device may be provided on a
single electrochemical test strip. The sensing device may not
require multiple discrete electrochemical test strips for
performing the simultaneous and multiplexed detection of the
plurality of target analytes.
[0022] In some embodiments, the plurality of semiconducting
nanostructures may comprise surfaces that are functionalized with a
linking reagent, and the capture reagents may be immobilized onto
the surfaces of the semiconducting nanostructures via the linking
reagent.
[0023] In some embodiments, the plurality of semiconducting
nanostructures may be thermally grown on said electrode(s) in a
configuration that aids in radial diffusion of the fluid sample
around the plurality of semiconducting nanostructures. The
plurality of semiconducting nanostructures may comprise, for
example ZnO nanostructures.
[0024] The fluid sample may be selected from the group consisting
of sweat, blood, serum, and urine of a human subject. In some
cases, the fluid sample may further include a room temperature
ionic liquid (RTIL) electrolyte buffer. The sensing device is
capable of determining the presence and concentration of the one or
more target analytes in a volume of the fluid sample equal to or
less than 30 .mu.L. In some embodiments, the substrate may comprise
a flexible and porous polyimide substrate having low absorption of
the fluid sample. The sensing device is capable of determining the
presence and concentration of the one or more target analytes,
without the use of any visual markers or labels conjugated to the
capture reagents.
[0025] In some embodiments, the plurality of semiconducting
nanostructures may be disposed on two or more electrodes comprising
a first electrode and a second electrode. A first capture reagent
may be attached to the semiconducting nanostructures on the first
electrode and configured to selectively bind to a first target
analyte. A second capture reagent may be attached to the
semiconducting nanostructures on the second electrode and
configured to selectively bind to a second target analyte. In some
embodiments, the first and second target analytes may comprise
different isoforms of a same type of biomarker. The sensing device
is capable of simultaneously determining the presence and
concentrations of the first and second target analytes upon binding
of the target analytes to the respective capture reagents. The
sensing device can be configured for both catalytic and
affinity-based detection of the one or more target analytes. In
some embodiments, the one or more target analytes may comprise a
plurality of cardiac biomarkers, and the plurality of capture
reagents may comprise a plurality of antibodies that are specific
to the plurality of cardiac biomarkers.
[0026] According to another aspect, a method of detecting one or
more target analytes in a fluid sample is provided. The method may
include providing a sensing device comprising (1) a substrate
comprising two or more electrodes, (2) a plurality of
semiconducting nanostructures disposed on at least one of said
electrodes, and (3) a plurality of capture reagents attached to the
plurality of semiconducting nanostructures. The method may also
include applying the fluid sample containing the one or more target
samples to the sensing device. The method may further include
detecting, with aid of sensing circuitry, changes to electron and
ion mobility and charge accumulation in different regions of the
semiconducting nanostructures and the fluid sample when the
plurality of capture reagents selectively bind to the one or more
target analytes in the fluid sample; and determining a presence and
concentration of the one or more target analytes based on the
detected changes to the electron and ion mobility and charge
accumulation.
[0027] A further aspect of the present disclosure is directed to a
sensing array for detecting a plurality of different target
analytes in a fluid sample. The array may comprise two or more
sensing devices disposed on a common substrate. The sensing devices
may each comprise a working electrode having a plurality of
semiconducting nanostructures disposed thereon and a capture
reagent attached to the semiconducting nanostructures. The fluid
sample may be applied to the electrodes of the two or more sensing
devices. The two or more sensing devices may comprise different
capture reagents that are configured to selectively bind to the
different target analytes in the fluid sample. The selective
binding is configured to effect changes to electron and ion
mobility and charge accumulation in different regions of the
semiconducting nanostructures and the fluid sample. Each of the
sensing devices can be configured to determine a presence and
concentration of a different target analyte in the fluid sample
based on detected changes to the electron and ion mobility and
charge accumulation. The changes can comprise simultaneous
modulation to the ion mobility in one or more regions adjacent to
the semiconducting nanostructures.
[0028] In some embodiments, the working electrodes of the two or
more sensing devices may have different types of semiconducting
nanostructures disposed thereon. In some cases, different types of
capture reagents may be attached to the different types of
semiconducting nanostructures.
[0029] In some embodiments, at least two of the sensing devices may
share a common reference electrode. Each of the at least two
sensing devices may further comprise a counter electrode. The
common reference electrode may be disposed between the working
electrodes of the at least two sensing devices. Additionally or
optionally, the common reference electrode may be disposed between
the counter electrodes of the at least two sensing devices.
[0030] In some embodiments, a first sensing device may comprise a
working electrode, a counter electrode and a reference electrode
located in proximity to each other in a first region of the
substrate. A second sensing device may comprise a working
electrode, a counter electrode and a reference electrode located in
proximity to each other in a second region of the substrate. The
first sensing device may comprise a first capture reagent
configured to selectively bind to a first target analyte, and the
second sensing device may comprise a second capture reagent
configured to selectively bind to a second target analyte. In some
embodiments, the first and second target analytes may be different
isoforms of a same type of biomarker.
[0031] In some embodiments, the electrodes of the two or more
sensing devices may be connected to sensing circuitry configured
for simultaneous acquisition and multiplexing of electrical signals
from the two or more sensing devices. The sensing circuitry can be
configured to analyze the electrical signals comprising of
impedance and capacitance signals. The signals may be indicative of
interfacial charge modulation comprising of the changes to the
electron and ion mobility. The signals may include capacitance
changes to space-charge regions formed in the semiconducting
nanostructures upon binding of the different target analytes to the
corresponding capture reagents.
[0032] In some embodiments, the sensing circuitry can be configured
to implement a plurality of electrochemical detection techniques
for detecting the impedance changes and the capacitance changes.
The plurality of electrochemical detection techniques may include a
modified EIS technique for measuring the impedance changes and
Mott-Schottky technique for measuring the capacitance changes. The
sensing array is capable of simultaneous and multiplexed detection
of the different target analytes present in the fluid sample using
the plurality of electrochemical detection techniques. The sensing
circuitry can be configured to perform the simultaneous and
multiplexed detection by analyzing the electrical impedance and
capacitance signals to determine the presence and concentration of
each of the different target analytes. The sensing circuitry can be
configured to perform the simultaneous and multiplexed detection
substantially in real-time upon binding of the different target
analytes to the corresponding capture reagents on the
semiconducting nanostructures.
[0033] The sensing circuitry can be configured to analyze the
impedance and capacitance signals by concurrently analyzing a set
of Nyquist plots obtained via the modified EIS technique and a set
of Mott-Schottky plots obtained via the Mott-Schottky technique. In
some embodiments, the modified EIS technique may comprise (1)
sectioning an interfacial charge layer for each of the two or more
sensing devices into a plurality of spatial dielectric z-planes
along a direction orthogonal to the interface between the fluid
sample and the semiconducting nanostructures, and (2) probing each
of the plurality of z-planes with a specific frequency selected
from a range of frequencies. Specific binding of different target
analytes to the corresponding capture reagents occurs at known
spatial heights within the plurality of interfacial charge layers
for the two or more sensing devices. The sensing circuitry can be
configured to determine the presence and concentration of each of
the different target analytes by measuring the capacitance and
impedance changes at specific frequencies corresponding to their
respective z-planes. The modified EIS technique is capable of
distinguishing the electrical impedance signals from background
noise at low concentrations of the different target analytes in the
fluid sample.
[0034] In some embodiments, the sensing array may be provided as a
single electrochemical test strip. The sensing array may not
require multiple discrete electrochemical test strips for
performing the simultaneous and multiplexed detection of the
different target analytes.
[0035] In some embodiments, the sensing circuitry can be configured
to selectively apply a plurality of modulation signals to the two
or more sensing devices to enable detection of the plurality of
different target analytes in the fluid sample. The sensing
circuitry can be configured to individually and selectively
control, activate, or modulate the two or more sensing devices. The
plurality of modulation signals can be configured to aid in
enhancing detection sensitivity of the different target
analytes.
[0036] A method of detecting a plurality of different target
analytes in a fluid sample is provided in accordance with another
aspect. The method may include providing the sensing array
disclosed herein; applying the fluid sample containing the one or
more target samples to the sensing array; and using each of the
sensing devices to determine the presence and concentration of a
different target analyte in the fluid sample, based on the detected
changes to the electron and ion mobility and charge accumulation in
the different regions of the semiconducting nanostructures and the
fluid sample.
[0037] A further aspect is directed to a sensor module for
detecting one or more target analytes in a fluid sample. The sensor
module may comprise a base module configured to releasably couple
to one or more discrete sensors. The one or more discrete sensors
can be used to determine a presence and concentration of the one or
more target analytes in the fluid sample based on detected changes
to electron and ion mobility and charge accumulation when the
discrete sensor(s) are coupled to the base module and the fluid
sample is applied to the sensor module. In some embodiments, the
sensor module may further comprise the one or more discrete
sensors.
[0038] The one or more discrete sensors can be configured to be
mechanically and electrically coupled to the base module. Each of
the one or more discrete sensors may comprise a working electrode
having a plurality of semiconducting nanostructures disposed
thereon and a capture reagent attached to the semiconducting
nanostructures. The base module may comprise at least one reference
electrode and at least one ground electrode. A plurality of
discrete sensors may comprise different capture reagents that are
configured to selectively bind to different target analytes in the
fluid sample. The selective binding is configured to effect changes
to the electron and ion mobility and charge accumulation in
different regions of the semiconducting nanostructures and the
fluid sample. The plurality of discrete sensors can be used for
determining the presence and concentration of the different target
analytes in the fluid sample.
[0039] In some embodiments, the base module may comprise (1) a
first receiving portion configured to couple to a first discrete
sensor, and (2) a second receiving portion configured to couple to
a second discrete sensor. The first discrete sensor may comprise a
first working electrode, and the second discrete sensor may
comprise a second working electrode. A first sensing device can be
formed by coupling the first discrete sensor to the first receiving
portion. The first sensing device may comprise the first working
electrode, a first counter electrode, and a reference electrode. A
second sensing device can be formed by coupling the second discrete
sensor to the second receiving portion. The second sensing device
may comprise the second working electrode, a second counter
electrode, and a reference electrode. In some embodiments, the
first sensing device and the second sensing device may share the
same reference electrode. The first sensing device can be
configured to determine the presence and concentration of a first
target analyte, and the second sensing device can be configured to
determine the presence and concentration of a second target
analyte.
[0040] In some embodiments, a method of using the sensor module for
detecting one or more target analytes in a fluid sample may
include: providing the base module that is configured to releasably
couple to one or more discrete sensors; coupling the one or more
discrete sensors to the base module thereby electrically and
mechanically connecting said discrete sensor(s) to the base module;
applying the fluid sample to the sensor module; and using the one
or more discrete sensors to determine a presence and concentration
of the one or more target analytes in the fluid sample based on
detected changes to electron and ion mobility and charge
accumulation that are specific to each of the one or more target
analytes.
[0041] In some embodiments, a method of using the sensor module for
detecting one or more target analytes in a fluid sample may
include: providing the base module that is configured to releasably
couple to one or more discrete sensors; coupling a first discrete
sensor to the base module thereby electrically and mechanically
connecting the first discrete sensor to the base module; applying
the fluid sample to the sensor module comprising the first discrete
sensor; and using the first discrete sensor to determine a presence
and concentration of a first target analyte in the fluid sample
based on detected changes to electron and ion mobility and charge
accumulation that are specific to the first target analyte. The
method may also comprise detaching the first discrete sensor from
the base module after the presence and concentration of the first
target analyte has been determined. The method may further comprise
coupling a second discrete sensor to the base module thereby
electrically and mechanically connecting the second discrete sensor
to the base module; applying the fluid sample to the sensor module
comprising the second discrete sensor; and using the second
discrete sensor to determine a presence and concentration of a
second target analyte in the fluid sample based on detected changes
to the electron and ion mobility and charge accumulation that are
specific to the second target analyte.
[0042] In some embodiments, a method of using the sensor module for
detecting two or more target analytes in a fluid sample may
include: providing the base module that is configured to releasably
couple to two or more discrete sensors; coupling a first discrete
sensor and a second discrete sensor to the base module thereby
electrically and mechanically connecting the first and second
discrete sensors to the base module; applying the fluid sample to
the sensor module comprising the first and second discrete sensors;
and (1) using the first discrete sensor to determine a presence and
concentration of a first target analyte in the fluid sample based
on detected changes to electron and ion mobility and charge
accumulation that are specific to the first target analyte, and (2)
using the second discrete sensor to determine a presence and
concentration of a second target analyte in the fluid sample based
on detected changes to the electron and ion mobility and charge
accumulation that are specific to the second target analyte. The
sensor module is capable of simultaneous and multiplexed detection
of the first and second target analytes present in the fluid sample
using a plurality of electrochemical detection techniques. The
plurality of electrochemical detection techniques may comprise (1)
a modified Electrochemical Impedance Spectroscopy (EIS) technique
for measuring impedance changes and (2) Mott-Schottky technique for
measuring capacitance changes.
[0043] In some embodiments, a kit for determining the presence and
concentration of one or more target analytes in a fluid sample may
include: a) a sensing device, a sensing array, and/or a sensor
module as described herein; and b) instructions for using the kit.
The kit may further comprise a diagnostic reader device or wearable
device configured to be in operable communication with the sensing
device, sensing array, and/or sensor module.
[0044] According to some aspects, a sensing apparatus or method may
be capable of simultaneously detecting (1) the presence and (2)
concentrations ranging from 0.1 to 10.sup.6 nGL with a coefficient
of variation less than 10%, of a plurality of different target
analytes in a single sample having a volume of less than 30 .mu.L.
The sensing apparatus or method may be capable of simultaneously
detecting the presence and concentrations of the plurality of
different target analytes in less than 2 minutes. The sensing
apparatus or method can be implemented using a single immunoassay
test strip. The sensing apparatus or method can be implemented
without using a separate immunoassay test strip to detect the
presence and concentration of each of the plurality of different
target analytes. The sensing apparatus or method is capable of
simultaneously detecting the presence and concentrations of the
plurality of different target analytes without the use of any
visually detectable markers or labels. The sensing apparatus or
method is capable of simultaneously detecting the presence and
concentrations of the plurality of different target analytes
comprising of (1) different biomarkers, (2) different isoforms of a
same type of biomarker, and/or (3) chemical agents. The sensing
apparatus or method can be implemented in a wearable device that is
worn on a portion of a user's body. Additionally or optionally, the
sensing apparatus or method can be implemented in a point-of-care
(POC) portable health diagnostics system. The sample may include
sweat, blood, serum, or urine of a human subject. The sample may be
provided with a room temperature ionic liquid (RTIL) electrolyte
buffer. The sensing apparatus or method is capable of
simultaneously detecting the presence and concentrations of the
plurality of different target analytes using catalytic and
affinity-based sensing mechanisms.
[0045] Additional aspects and advantages of the present disclosure
will become readily apparent to those skilled in this art from the
following detailed description, wherein only illustrative
embodiments of the present disclosure are shown and described. As
will be realized, the present disclosure is capable of other and
different embodiments, and its several details are capable of
modifications in various obvious respects, all without departing
from the disclosure. Accordingly, the drawings and description are
to be regarded as illustrative in nature, and not as
restrictive.
INCORPORATION BY REFERENCE
[0046] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0048] FIG. 1 shows a schematic of a sensing device in accordance
with some embodiments;
[0049] FIG. 2 shows a sensing array comprising a plurality of
sensing devices for detecting different target analytes;
[0050] FIG. 3 shows a multi-configurable sensing array comprising a
plurality of sensing devices configured for simultaneous and
multiplexed detection of a plurality of target analytes;
[0051] FIG. 4 shows a multi-configurable sensing array in
accordance with some embodiments;
[0052] FIG. 5 shows a multi-configurable sensing system in
accordance with some embodiments;
[0053] FIGS. 6A-6C show an SEM micrograph and ATR-FTIR spectra of
ZnO nanostructures selectively grown on a working electrode, in
accordance with some embodiments;
[0054] FIGS. 7A-7D show the functionalization of a working
electrode in accordance with some embodiments;
[0055] FIGS. 8A-8D show fluid sample absorption onto different
working electrodes and z-plane fragmentation using a modified EIS
technique;
[0056] FIGS. 9A-9D show electrical simulation results for the
sensing array of FIG. 5;
[0057] FIGS. 10A and 10B show the baseline electrochemical response
of the sensing array of FIG. 5, and the impedance response at each
step of the immunoassay;
[0058] FIGS. 11A-11D show Nyquist plots and calibration curves
representing the detection of cTnI and cTnT using the sensing array
of FIG. 5;
[0059] FIGS. 12A-12D show Mott-Schottky capacitance and calibration
curves plotted as a function of applied potential for cTnI and cTnT
detection using the sensing array of FIG. 5;
[0060] FIG. 13A shows a calibration curve representing the
detection of NT-proBNP using the sensing array of FIG. 5;
[0061] FIG. 13B shows the correlation between NT-proBNP detection
using an exemplary sensing array and NT-proBNP detection using a
conventional enzyme-linked immunosorbent assay (ELISA);
[0062] FIG. 14 shows a sensing platform comprising a test strip and
a diagnostic reader device, in accordance with some
embodiments;
[0063] FIG. 15 shows a sensing platform comprising a wearable
device in accordance with some embodiments;
[0064] FIG. 16 is a flowchart showing a method for continuous,
real-time detection of alcohol, EtG, and EtS in accordance with
some embodiments.
[0065] FIGS. 17A-17F show different electrical field simulations
for a multi-configurable sensing array comprising a plurality of
electrodes; and
[0066] FIGS. 18A-C show a modular sensing device in accordance with
some embodiments; and
[0067] FIGS. 19A and 19B show a multi-configurable modular sensing
array in accordance with some embodiments.
DETAILED DESCRIPTION
[0068] Reference will now be made in detail to exemplary
embodiments of the disclosure, examples of which are illustrated in
the accompanying drawings. Wherever possible, the same reference
numbers will be used throughout the drawings and disclosure to
refer to the same or like parts.
[0069] The following is an overview of the contents in this
disclosure:
[0070] I. General
[0071] II. Sensing Device [0072] A. Substrate [0073] B. Electrodes
[0074] C. Semiconducting Nanostructures [0075] D. Capture Reagents
[0076] E. Test Zone [0077] F. Sample and Target Analytes [0078] G.
Sensing Mechanisms [0079] H. Room-Temperature Ionic Liquids
(RTIL)
[0080] III. Multi-configurable Sensing Array [0081] A. Simultaneous
and Multiplexed Detection of Multiple Target Analytes [0082] B.
Electrode Configurations
[0083] IV. Sensing System [0084] A. Multiplexer and Sensing
Circuitry [0085] B. Modified EIS [0086] C. Simulation and Design
[0087] D. Baseline Characterization [0088] E. Electrochemical
Signal Responses
[0089] V. Sensing Platforms [0090] A. Diagnostics Reader Device
[0091] B. Wearable Device
[0092] VI. Modular Sensing Device/Array
[0093] VII. Kits
[0094] Provided herein are sensing devices, arrays of devices, and
methods of using the same. Also provided herein are systems and
devices configured to receive and analyze signals from the sensing
devices or arrays, and provide an output based on the sensing
results. Further provided herein are kits comprising modular
sensing devices and arrays.
[0095] The various embodiments described herein may be useful for
performing immunoassay tests on a sample, for example, to diagnose
a disease or to provide information regarding a biological state or
condition of a subject. The disclosed devices, arrays, systems,
methods, and kits may be useful for detecting the presence and
concentration of a wide variety of analytes in a sample. In many
cases, the disclosed embodiments can enable simultaneous and
multiplexed detection of the presence and concentration of multiple
analytes in a single sample, via a common sensing platform. The
various embodiments described herein are capable of detecting the
presence and concentration of more than one analyte in a sample
with greater specificity and/or sensitivity than currently
available sensing devices or immunoassays. In many cases, the
devices, arrays, systems, methods, and kits provided herein can
enable a user to perform quantitative measurements with higher
accuracy and wider dynamic range than currently available sensing
devices or immunoassays.
[0096] As used in the specification and claims, the singular form
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a cell" includes
a plurality of cells, including mixtures thereof.
[0097] As used in the specification and claims, the term
"apparatus" may include a device, an array of devices, a system,
and any embodiments of the sensing applications described
herein.
[0098] As used herein, the term "about" a number refers to that
number plus or minus 10% of that number. The term "about" a range
refers to that range minus 10% of its lowest value and plus 10% of
its greatest value.
[0099] I. General
[0100] Presently, there is a need for multiplexed immunoassays that
can be used for simultaneous detection of multiple analytes in a
short period of time, from a small sample volume, and at reduced
costs. A key challenge lies in quantitative detection of biomarkers
in a simultaneous or multiplexed manner at the early stages of a
disease, especially if the sample contains very low concentrations
of the biomarkers. To address this challenge, accuracy in diagnosis
of the disease can be enhanced by quantification through a panel of
biomarkers indicative or associated with the disease. Accordingly,
there is interest and value in designing ultrasensitive sensing
devices that are capable of detection of a panel of biomarkers from
a single sample of human body fluids.
[0101] A number of transduction mechanisms can be used to achieve
ultra-sensitive and multiplexed label-free biomarker detection. An
example of such transduction mechanisms may include
electrical/electrochemical-based sensing platforms, which typically
involve capturing biomarkers on the surface of electrode materials.
This phenomenon transduces the biological signal into a measurable
electrical signal response, which can then be used to detect the
presence and concentration of the biomarker in the sample. The
structural and morphological characteristics of the electrode
materials play an important role in achieving both sensitivity and
selectivity required for ultrasensitive biomarker detection.
Precise control over size and shape of the materials on a nanoscale
level can yield nanostructures with enhanced chemical and physical
properties, that can be tailored towards the design of robust
ultrasensitive sensing platforms. For example, the availability of
a large number of surface atoms in extended (out-of-plane)
nanostructures can allow amplification of a biological signal
response, when compared to their planar sensing electrode
counterparts, thereby enabling improved sensing
characteristics.
[0102] Detection of analytes can be based upon enzymatic sensing
devices for the detection of glucose, cholesterol, lactic acid,
uric acid, etc. Quantification of such analytes may be based upon
detection of byproducts of enzymatic reactions where non-specific
interactions may be an issue. Technological bottlenecks associated
with non-specific interactions can be minimized by use of specific
capture probes. For example, affinity-based sensing mechanisms for
designing immunoassay-based sensing devices using non-faradic
approaches can be used. In some cases, semiconducting
nanostructures can be used to facilitate direct electron transport
as their electrical properties are strongly altered by charge
perturbations occurring due to biomolecular confinement and binding
events. The electrical detection/sensing methods described herein
can permit direct characterization of capture probe--target
biomarker interaction, based on charge perturbations at the
electrode/electrolyte interface.
[0103] When an electrode comprising nanostructures on its surface
is exposed to an ionic solution containing biomolecules, a
potential difference can be created at the electrode/electrolyte
interface due to unequal distribution of charges. As a result of
biomolecular binding events at the nanostructured electrode
surface, redistribution of charges in the electrode and ions in the
electrolyte can result in formation of a space-charge region within
the nanostructures and at an electrical double layer at the
electrode/electrolyte interface. Biomarker binding can be evaluated
and quantified by measuring changes in electrode impedance and/or
capacitance at selected frequencies. In some embodiments, changes
to the space-charge capacitance and overall impedance at the
electrode/electrolyte interface can be measured using both
Mott-Schottky technique and a modified electrochemical impedance
spectroscopy (EIS) technique which are described in detail herein.
A correlation in output signal response with concentration can be
determined between (and using) both detection techniques, which
provide a combinatorial approach for the accurate and sensitive
detection of protein biomarkers.
[0104] The electrochemical sensing devices, arrays and methods
described herein can be used for detecting multiple biomarkers. The
sensing devices and arrays can be designed and fabricated on
various substrates. The substrates may be rigid or flexible.
Examples of suitable substrates may include silicon, glass, printed
circuit boards, polyurethane, polycarbonate, polyamide, polyimide,
and the like. The sensing devices and arrays can be used for
continuous and real-time detection, monitoring, and quantification
of various chemical and biological agents in body fluids. Examples
of body fluids may include blood, sweat, tears, urine, saliva, and
the like. Real-time detection can be performed in a single-use or
in a continuous-use manner using the sensing technology platform
described herein. The challenges of multiplexed detection of
specific proteins can be addressed by the present inventions, which
are directed to: (1) the designs of a microelectrode sensor
platform comprising an array of multi-configurable sensing device
each independently functionalized for specific detection of a
target biomarker(s), and (2) each sensor output/results being
independently measured and transduced to provide a combinatorial
outcome relating to the end physiological state being
predicted.
[0105] An important aspect in affinity-based sensing devices
relates to the specificity of the sensor. The term "specificity"
may be described as the ability of the sensor to respond
specifically to target biomolecules, but not to other similar
biomolecules. Generally, current electrical-based label-free
sensing devices are often unable to distinguish between specific
and nonspecific interactions except via probe specificity,
regardless of the readout method. Specificity is often important
for detection of biomolecules in real-world samples such as blood,
serum, urine, saliva, sweat, etc., where the target concentration
can be much lower than the concentration of non-target biomolecules
present in the samples. For instance, blood serum typically
contains around 70 mg/mL total protein content; however, disease
biomarker proteins may be expressed in concentrations in the lower
pg/mL regime. Thus, a sensing device that can detect 1 pg/mL of the
protein in a saline solution but manifests a 1 ng/mL response in
blood, may not be useful in a clinical setting unless the serum is
depleted of interfering plasma proteins, or if some other
compensations were made.
[0106] In the various embodiments described herein, specificity to
the detection of target biomarkers, within each sensor on the
platform array, can be achieved through specific antibody
immobilization on microelectrode surfaces having semiconducting
nanostructures (e.g. ZnO), functionalized using thiol-based and/or
phosphonic-based linker chemistries to achieve stable and robust
immobilization of the proteins. Target protein specific monoclonal
antibodies can be introduced onto the linker functionalized
nanostructured ZnO surfaces in the presence of a room temperature
ionic liquid (RTIL) electrolyte buffer. The properties of the RTIL
can be adjusted to ensure long term stability (prevent denaturing
of the protein antibody from pH, temperature and environment), and
enhance the efficacy in selective binding to the nanostructured ZnO
surfaces. A modified electrochemical impedance spectroscopy (EIS)
technique as described herein can be used for enabling
ultra-sensitive and highly-specific detection of proteins.
[0107] Examples of biosensing systems and methods are described in
U.S. Patent Application Publication No. 2016/146754; U.S.
Provisional Application Nos. 62/554,841 and 62/554,956; non-patent
literature "Ultrasensitive and low-volume point-of-care diagnostics
on flexible strips--a study with cardiac troponin biomarkers,"
Nandhinee Radha Shanmugam, Sriram Muthukumar, and Shalini Prasad,
Nature, Scientific Reports 6, Article Number 33423, (2016); and "A
wearable biochemical sensor for monitoring alcohol consumption
lifestyle through Ethyl glucuronide (EtG) detection in human
sweat," Anjan Panneer Selvam, Sriram Muthukumar, Vikramshankar
Kamakoti, and Shalini Prasad, Nature, Scientific Reports 6, Article
number: 23111 (2016), the entire contents of which are herein
incorporated by reference.
[0108] II. Sensing Device
[0109] Disclosed herein is a sensing device for detecting one or
more target analytes in a fluid sample. The sensing device may
include a substrate comprising two or more electrodes. A plurality
of semiconducting nanostructures may be disposed on at least one of
the electrodes. A plurality of capture reagents may be attached to
the plurality of semiconducting nanostructures. The plurality of
capture reagents are configured to selectively bind to the one or
more target analytes in the fluid sample, thereby effecting changes
to electron and ion mobility and charge accumulation in different
regions of the semiconducting nanostructures and the fluid sample.
The changes to the electron and ion mobility and charge
accumulation are detectable with aid of sensing circuitry, and can
be used to determine a presence and concentration of the one or
more target analytes in the fluid sample.
[0110] Embodiments of the present disclosure are also directed to a
method of detecting one or more target analytes in a fluid sample.
The method may include providing a sensing device comprising (1) a
substrate comprising two or more electrodes, (2) a plurality of
semiconducting nanostructures disposed on at least one of said
electrodes, and (3) a plurality of capture reagents attached to the
plurality of semiconducting nanostructures. The method may include
applying the fluid sample containing the one or more target samples
to the sensing device. Additionally, the method may include
detecting, with aid of sensing circuitry, changes to electron and
ion mobility and charge accumulation in different regions of the
semiconducting nanostructures and the fluid sample when the
plurality of capture reagents selectively bind to the one or more
target analytes in the fluid sample. The method may further include
determining a presence and concentration of the one or more target
analytes based on the detected changes to the electron and ion
mobility and charge accumulation.
[0111] FIG. 1 shows a schematic of a sensing device 100 in
accordance with some embodiments. The sensing device 100 may be
used to conduct one or more immunoassays for detecting one or more
target analytes in a sample. The sensing device may contain a
plurality of capture reagents for conducting the one or more
immunoassays. The capture reagents may be disposed or immobilized
on a surface of at least one electrode of the sensing device.
Generally, the sensing device comprises materials suitable for
performing biosensing, by providing appropriate materials for
immobilizing or otherwise providing various capture reagents to
perform the immunoassay.
[0112] A. Substrate
[0113] Referring to FIG. 1, the sensing device 100 may comprise a
substrate 110. The substrate may be flexible or rigid. The
substrate may include materials such as polyimide, silicon, glass,
printed circuit boards (PCB), polyurethane, polycarbonate,
polyamide, or the like. In some embodiments, the substrate may be
an organic substrate comprising flexible PCB materials. In some
embodiments, the substrate may be a flexible and porous polyimide
substrate that allows very low volumes of fluid adsorption within
its pores, which in turn facilitates more effective conjugation and
thus improved sensitivity in the detection of one or more target
analytes present in the fluid sample. In some embodiments, the
substrate may be capable of flexing or bending a large number of
cycles without substantially impacting the accuracy and sensitivity
of the sensing device.
[0114] In some embodiments, the substrate may comprise test strips
for aiding lateral transport of a sample fluid to electrodes on the
sensing device. Non-limiting examples of test strips may include
porous paper, or a membrane polymer such as nitrocellulose,
polyvinylidene fluoride, nylon, Fusion 5.TM., or
polyethersulfone.
[0115] In some embodiments, the sensing device may be provided on a
single electrochemical test strip. For example, the sensing device
need not include multiple electrochemical test strips for
performing the simultaneous and multiplexed detection of a
plurality of target analytes.
[0116] B. Electrodes
[0117] The sensing device 100 may comprise two or more electrodes
disposed on the substrate. For example, in the embodiment shown in
FIG. 1, a working electrode (WE) 120, a reference electrode (RE)
130, and a counter electrode (CE) 140 may be disposed on the
substrate 110. Any number or type of electrodes may be
contemplated. The electrodes may be exposed to a sample suspected
to contain one or more target analytes. A working electrode (WE) as
described anywhere herein may be referred to interchangeably as a
sensing electrode, a sensing working electrode, detection
electrode, or the like. The WE 120 may comprise a conducting
electrode stack. The WE 120 may further comprise a semiconducting
sensing element (e.g., a plurality of semiconducting nanostructures
122) formed on its surface, as described in detail elsewhere
herein. The RE 130 and CE 140 may each comprise a conducting
electrode stack, and need not comprise sensing elements on their
surfaces. For example, the RE 130 and CE 140 need not include
molecules that are used for functionalizing the sensing element on
the WE 120. The CE 140 and RE 130 may be electrochemically
inert/stable, and may collectively form an electrochemical cell
with the WE 120 when the electrodes come into contact with the
fluid sample (electrolyte or ionic liquid).
[0118] The electrodes may be formed of various shapes and/or sizes.
The electrodes may have a substantially circular or oval shape, for
example as shown in FIG. 1. In some embodiments, the electrodes may
have a regular shape (e.g. polygonal shapes such as triangular,
pentagonal, hexagonal, etc.) or an irregular shape. The electrodes
may be of the same size or different sizes. The electrodes may have
the surface areas or different surface areas. The ratio of the
surface areas of WE:CE:RE may be given by x:y:z, where x, y and z
may be any integer. In some instances, z may be larger than x and
y, such that the RE 130 has a larger surface area than each of WE
120 and CE 140. For example, the ratio of the surface areas of
WE:CE:RE may be 1:1:2, 1:1:3, 1:1:4, 1:1:5, 1:1:6, or any other
ratio. In some preferred embodiments, the ratio of the surface
areas of WE:CE:RE may be 1:1:4, but is not limited thereto.
[0119] The electrodes on the sensing device 100 may be electrically
connected to a plurality of contact pads via conducting layer
traces 102 embedded or formed on the substrate. Each electrode may
be connected to a contact pad. For example, the working electrode
120 may be connected to a first contact pad 121, the reference
electrode 130 may be connected to a second contact pad 131, and the
counter electrode 140 may be connected to a third contact pad 141.
In some alternative embodiments, two or more electrodes may be
connected to a contact pad. Optionally, an electrode may be
connected to two or more contact pads. The contact pads may be
located at a distance from the electrodes. In some embodiments, the
contact pads and electrodes may be located at opposite ends of the
substrate. The contact pads may be provided on a same surface of
the substrate 110 as the electrodes. Alternatively, the contact
pads may be provided on a different surface of the substrate 110 as
the electrodes. For example, the contact pads and the electrodes
may be provided on opposite surfaces of the substrate.
[0120] The conducting layer traces 102 may be formed of a metal,
e.g. Cu. The electrodes 120, 130, and 140 may include a surface
finish formed on the conducting layer traces. Non-limiting examples
of surface finishes may include electroless nickel deposited on a
copper trace, or an immersion gold/immersion silver/electrolytic
gold deposited on an electroless nickel surface.
[0121] In some embodiments, different surface finishes on a
flexible printed circuit board substrate may comprise the following
exemplary thickness ranges: (1) For Immersion Silver, 8-15
micro-inches of 99% pure silver over Cu trace layer with good
surface planarity, which may be a preferred surface finish for RE
130. In some cases, the post immersion silver surface finish may be
chemically modified to form an Ag/AgCl surface that offers
excellent electrochemical stability. (2) For Electroless Nickel
Immersion Gold (ENIG), 2-8 micro-inches Au layer over 120-240
micro-inches electroless Ni layer over Cu trace layer. (3) For
Electroless Nickel Electroless Palladium Immersion Gold (ENEPIG),
2-8 micro-inches Au layer over 4-20 micro-inches electroless Pd
layer over 120-240 micro-inches electroless Ni layer. The Pd layer
can eliminate corrosion potential from immersion reaction. Au
surfaces are relatively stable/inert, offer wide electrochemical
window and can be used for the WE 120 and CE 140. It should be
appreciated that the above thickness values are merely exemplary,
and that different thickness values may be contemplated for
different surface finishes depending on the desired electrical and
sensing properties.
[0122] C. Semiconducting Nanostructures
[0123] Semiconducting nanostructures may be disposed on at least
one of the electrodes to aid in sensing of one or more target
analytes. For example, a sensing element comprising a layer of
semiconducting nanostructures 122 may be deposited over the surface
of the WE 120. The WE 120 may include one or more of the surface
finishes described herein. The choice of semiconducting
nanostructures 122 may be determined based on the catalytic
properties of the semiconducting material. In some embodiments,
metal oxide nanostructured surfaces can offer immobilization when
selectively functionalized with thiol and phosphonic acid linker
chemistries to form specific interactions with the protein
biomolecules, that can lead to enhancements in specific output
signal response and enhanced specificity in biomarker
detection.
[0124] Non-limiting examples of semiconducting materials that can
be used on a working electrode may include the following: Diamond,
Silicon, Germanium, Gray tin (.alpha.-Sn), Sulfur (.alpha.-S), Gray
selenium, Tellurium, Silicon carbide (3C--SiC), Silicon carbide
(4H--SiC), Silicon carbide (6H--SiC), Boron nitride (cubic), Boron
nitride (hexagonal), Boron nitride (nanotube), Boron phosphide,
Boron arsenide, Aluminium nitride, Aluminium phosphide, Aluminium
arsenide, Aluminium antimonide, Gallium nitride, Gallium phosphide,
Gallium, arsenide, Gallium antimonide, Indium nitride, Indium,
phosphide, Indium arsenide, Indium antimonide, Cadmium selenide,
Cadmium, sulfide, Cadmium telluride, Zinc oxide, Zinc selenide,
Zinc sulfide, Zinc telluride, Cuprous, chloride, Copper sulfide,
Lead selenide, Lead(II) sulfide, Lead telluride, Tin sulfide, Tin
sulfide, Tin telluride, Bismuth, telluride, Cadmium phosphide,
Cadmium arsenide, Cadmium antimonide, Zinc phosphide, Zinc
arsenide, Zinc antimonide, Titanium dioxide (anatase), Titanium
dioxide (rutile), Titanium dioxide (brookite), Copper(I) oxide,
Copper(II) oxide, Uranium, dioxide, Uranium, trioxide, Bismuth,
trioxide, Tin dioxide, Lead(II) iodide, Molybdenum disulfide,
Gallium, selenide, Tin sulfide, Bismuth sulfide, Iron(II) oxide,
Nickel(II) oxide, Europium(II) oxide, Europium(II) sulfide,
Chromium(III) bromide, Arsenic sulfideOrpiment, Arsenic
sulfideRealgar, Platinum, silicide, Bismuth(III) iodide,
Mercury(II) iodide, Thallium(I) bromide, Silver sulfide, Iron
disulfide, Lead tin, telluride, Thallium tin telluride, Thallium
germanium telluride, Barium titanate, Strontium, titanate, Lithium
niobate, Lanthanum copper oxide, Gallium manganese arsenide, Indium
manganese arsenide, Cadmium manganese telluride, Lead manganese
telluride, Copper indium selenide (CIS), Silver gallium sulfide,
Zinc silicon phosphide, Copper tin sulfide (CTS), Lanthanum calcium
manganite, Copper zinc tin sulfide (CZTS), or Copper zinc antimony
sulfide (CZAS).
[0125] Non-limiting examples of semiconductor alloy materials that
can be used on a working electrode may include the following:
Silicon-germanium, Silicon-tin, Aluminium gallium arsenide, Indium
gallium arsenide, Indium gallium phosphide, Aluminium indium
arsenide, Aluminium indium antimonide, Gallium arsenide nitride,
Gallium arsenide phosphide, Gallium arsenide antimonide, Aluminium
gallium nitride, Aluminium gallium phosphide, Indium gallium
nitride, Indium arsenide antimonide, Indium gallium antimonide,
Cadmium zinc telluride (CZT), Mercury cadmium telluride, Mercury
zinc telluride, Mercury zinc selenide, Aluminium gallium indium
phosphide, Aluminium gallium arsenide phosphide, Indium gallium
arsenide phosphide, Indium gallium arsenide antimonide, Indium
arsenide antimonide phosphide, Aluminium indium arsenide phosphide,
Aluminium gallium arsenide nitride Indium gallium arsenide nitride,
Indium aluminium arsenide nitride, Gallium arsenide antimonide
nitride, Copper indium gallium selenide (CIGS), Gallium indium
nitride arsenide antimonide, or Gallium indium arsenide antimonide
phosphide.
[0126] In some preferred embodiments, the plurality of
semiconducting nanostructures 122 may comprise ZnO. ZnO is suitable
for detecting biomolecules for a wide range of disease biomarkers
due to its multifunctional characteristics and ability to form
anisotropic nanostructures. The properties of ZnO such as good
biocompatibility, wide band gap, non-toxicity, fast electron
transfer, high isoelectricpoint (IEP: 9.5), favorable surface for
linker chemistry binding, ease in formation of highly c-axis
oriented nanostructures at low temperatures (<100.degree. C.)
and on various substrates including flexible polymeric substrates,
and heightened sensitivity to adsorbed molecules render ZnO an
attractive material of choice for affinity sensing applications and
with both direct current (DC) and alternating current (AC)
electrochemical methods. ZnO is preferred for designing sensors
based on electrical transduction. Furthermore, ZnO with its single
crystalline state is advantageous in the integration with flexible
polymeric substrates, and offers low-cost of ownership
manufacturing processes.
[0127] It is noted that any semiconducting materials with
appropriate functionalization can be utilized on the working
electrode(s) of the sensing device. In some embodiments, the metal
oxide thin films and nanostructures of ZnO, TiO.sub.2,
CNT-TiO.sub.2, SnO.sub.2, ZrO.sub.2, etc. can be used for design of
glucose oxide, cholesterol oxidase and other enzymatic sensing
devices. For catalytic based sensing devices, the choice of
metal/semiconductor (examples: Ag, Au, Pd, Ni, Zn, Co, W, Mo, Mn,
and their respective alloys such as ZnO, TiO.sub.2, MnO.sub.2,
MoS.sub.2, etc.) as the sensing electrode material may also be
dependent on the electrocatalytic properties of the material and
the stability of the material at the temperature of operation of
the sensor, the pH range of the buffer solution containing the
target analytes, and the electrochemical potential window for the
detection of the target analytes.
[0128] In some embodiments, the plurality of semiconducting
nanostructures 122 may be thermally grown on the working electrode
in a configuration that aids in radial diffusion of the sample
around the plurality of semiconducting nanostructures. As an
example, the formation of ZnO nanostructures is described in detail
with reference to FIGS. 6A-6C.
[0129] D. Capture Reagents
[0130] A plurality of capture reagents 124 may be attached to the
plurality of semiconducting nanostructures 122 on the surface of
the working electrode 120. The plurality of capture reagents are
configured to selectively bind to one or more target analytes in a
fluid sample, thereby effecting changes to electron and ion
mobility and charge accumulation in different regions of the
semiconducting nanostructures and the fluid sample. The changes to
the electron and ion mobility and charge accumulation are
detectable with aid of sensing circuitry, and can be used to
determine a presence and concentration of the one or more target
analytes in the fluid sample.
[0131] The capture reagents 124 may include an antibody or antibody
fragment, an antigen, an aptamer, a peptide, a small molecule, a
ligand, a molecular complex or any combination thereof.
Essentially, the capture reagents may be any reagents that have
specific binding activity for different target analytes. In some
cases, a first capture reagent and a second capture reagent may be
antibodies or antibody fragments that specifically bind to epitopes
present on a first target analyte and a second target analyte,
respectively. Immunoglobulin molecules can be of any type (e.g.,
IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3,
IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. In
some cases, the antibody is an antigen-binding antibody fragment
such as, for example, a Fab, a F(ab'), a F(ab')2, a Fd chain, a
single-chain Fv (scFv), a single-chain antibody, a disulfide-linked
Fv (sdFv), a fragment comprising either a VL or VH domain, or
fragments produced by a Fab expression library. Antigen-binding
antibody fragments, including single-chain antibodies, can comprise
the variable region(s) alone or in combination with the entirety or
a portion of the following: hinge region, CH1, CH2, CH3 and CL
domains. Also, antigen-binding fragments can comprise any
combination of variable region(s) with a hinge region, CH1, CH2,
CH3 and CL domains. Antibodies and antibody fragments may be
derived from a human, rodent (e.g., mouse and rat), donkey, sheep,
rabbit, goat, guinea pig, camelid, horse, or chicken. Various
antibodies and antibody fragments may be designed to selectively
bind essentially any desired analyte. Methods of generating
antibodies and antibody fragments are well known in the art.
[0132] The terms "selective" or "specific" binding may be used
herein interchangeably. Generally speaking, a ligand that
selectively or specifically binds to a target means that the ligand
has a high binding affinity for its target, and a low binding
affinity for non-target molecules. The dissociation constant
(K.sub.d) may be used herein to describe the binding affinity of a
ligand for a target molecule (e.g., an analyte). The dissociation
constant may be defined as the molar concentration at which half of
the binding sites of a target molecule are occupied by the ligand.
Therefore, the smaller the K.sub.d, the tighter the binding of the
ligand to the target molecule. In some cases, a ligand has a
dissociation constant (K.sub.d) for a target molecule of less than
1 mM, less than 100 .mu.M, less than 10 .mu.M, less than 1 .mu.M,
less than 100 nM, less than 50 nM, less than 25 nM, less than 10
nM, less than 5 nM, less than 1 nM, less than 500 .mu.M, less than
100 .mu.M, less than 50 .mu.M, or less than 5 .mu.M.
[0133] The plurality of semiconducting nanostructures may comprise
surfaces that are functionalized with a linking reagent. The
capture reagents may be immobilized onto the surfaces of the
semiconducting nanostructures via the linking reagent, which is
described in detail with reference to FIGS. 7A-7D.
[0134] The sensing device is capable of determining the presence
and concentration of one or more target analytes in a sample,
without the use of any visual markers or labels conjugated to the
capture reagents. In various embodiments, the capture detection
reagents need not be conjugated or otherwise attached to a
detectable label. A detectable label may be a fluorophore, an
enzyme, a quencher, an enzyme inhibitor, a radioactive label, one
member of a binding pair or any combination thereof. In contrast,
other known protein sensing devices often require a label attached
to the target protein for detection and quantification. Labeling a
biomolecule can drastically change its binding properties, and the
yield of the target-label coupling reaction can be highly variable
which may affect the detection of protein targets.
[0135] The sensing device disclosed herein can circumvent the
issues associated with labeling, by using label-free methods for
protein detection. Many protein sensors are affinity-based which
uses an immobilized capture reagent that binds a target
biomolecule. The challenge of detecting a target analyte in
solution lies in detecting changes at a localized surface. The use
of nanomaterials (e.g. semiconducting nanostructures) as capture
surfaces can be particularly beneficial when designing
ultra-sensitive electrical sensing devices that rely on measured
current and/or voltage to detect binding events. Electrical sensing
techniques, such as the modified electrochemical impedance
spectroscopy (EIS) technique described herein, have the ability to
rapidly detect protein biomarkers at low concentrations. Impedance
measurements can be especially useful since they do not require
special labels and are therefore suitable for label-free capture
operation.
[0136] E. Test Zone
[0137] Referring to FIG. 1, the substrate 110 may include a test
zone 150 for receiving a sample. The test zone may correspond to a
portion or region of the sensing device that is configured to
receive or accept a sample. The test zone may be located anywhere
on the sensing device, for example at or near an end portion of the
substrate. A sample may be applied to the test zone by, e.g.,
inserting the end portion of the device containing the test zone
into a container holding the sample, by pipetting a fluid sample
directly onto the test zone, or by holding the test zone of the
device under a fluid stream. Generally, the sample is a fluid
sample. In other cases, the sample is a solid sample that is
modified to form a fluid sample, for example, dissolved or
disrupted (e.g., lysed) in a liquid medium.
[0138] In some embodiments, a test zone may optionally include a
pad or other contact surface. In some cases, the pad may be
composed of a woven mesh or a fibrous material such as a cellulose
filter, polyesters, or glass fiber. The test zone may further
include, without limitation, pH and ionic strength modifiers such
as buffer salts (e.g., Tris), viscosity enhancers to modulate flow
properties, blocking and resolubilization agents (e.g., proteins
(such as albumin), detergents, surfactants (such as Triton X-100,
Tween-20), and/or filtering agents (e.g., for whole blood)).
[0139] F. Sample and Target Analytes
[0140] Generally, the sample applied to the test zone 150 may be a
fluid sample or a solid sample modified with a liquid medium. In
various aspects, the sample is a biological sample. Non-limiting
examples of biological samples suitable for use with the
immunoassay devices of the disclosure include: whole blood, blood
serum, blood plasma, urine, feces, saliva, vaginal secretions,
semen, interstitial fluid, mucus, sebum, sweat, tears, crevicular
fluid, aqueous humour, vitreous humour, bile, breast milk,
cerebrospinal fluid, cerumen, enolymph, perilymph, gastric juice,
peritoneal fluid, vomit, and the like. The biological sample can be
obtained from a hospital, laboratory, clinical or medical
laboratory. In some cases, the immunoassay test using the sensing
device is performed by a clinician or laboratory technician. In
other cases, the immunoassay test using the sensing device is
performed by the subject, for example, at home.
[0141] The biological sample can be from a subject, e.g., a plant,
fungi, eubacteria, archaebacteria, protist, or animal. The subject
can be an organism, either a single-celled or multi-cellular
organism. The subject can be cultured cells, which can be primary
cells or cells from an established cell line, among others.
Examples of cell lines include, but are not limited to, 293-T human
kidney cells, A2870 human ovary cells, A431 human epithelium, B35
rat neuroblastoma cells, BHK-21 hamster kidney cells, BR293 human
breast cells, CHO Chinese hamster ovary cells, CORL23 human lung
cells, HeLa cells, or Jurkat cells. The sample can be isolated
initially from a multi-cellular organism in any suitable form. The
animal can be a fish, e.g., a zebrafish. The animal can be a
mammal. The mammal can be, e.g., a dog, cat, horse, cow, mouse,
rat, or pig. The mammal can be a primate, e.g., a human,
chimpanzee, orangutan, or gorilla. The human can be a male or
female. The sample can be from a human embryo or human fetus. The
human can be an infant, child, teenager, adult, or elderly person.
The female can be pregnant, suspected of being pregnant, or
planning to become pregnant. The female can be ovulating. In some
cases, the sample is a single or individual cell from a subject and
the biological sample is derived from the single or individual
cell. In some cases, the sample is an individual micro-organism, or
a population of micro-organisms, or a mixture of micro-organisms
and host cells.
[0142] In some cases, the biological sample comprises one or more
bacterial cells. In some cases, the one or more bacterial cells are
pathogens. In some cases, the one or more bacterial cells are
infectious. Non-limiting examples of bacterial pathogens that can
be detected include Mycobacteria (e.g. M. tuberculosis, M. bovis,
M. avium, M. leprae, and M. africanum), rickettsia, mycoplasma,
chlamydia, and legionella. Some examples of bacterial infections
include, but are not limited to, infections caused by Gram positive
bacillus (e.g., Listeria, Bacillus such as Bacillus anthracis,
Erysipelothrix species), Gram negative bacillus (e.g., Bartonella,
Brucella, Campylobacter, Enterobacter, Escherichia, Francisella,
Hemophilus, Klebsiella, Morganella, Proteus, Providencia,
Pseudomonas, Salmonella, Serratia, Shigella, Vibrio and Yersinia
species), spirochete bacteria (e.g., Borrelia species including
Borrelia burgdorferi that causes Lyme disease), anaerobic bacteria
(e.g., Actinomyces and Clostridium species), Gram positive and
negative coccal bacteria, Enterococcus species, Streptococcus
species, Pneumococcus species, Staphylococcus species, and
Neisseria species. Specific examples of infectious bacteria
include, but are not limited to: Helicobacter pyloris, Legionella
pneumophilia, Mycobacterium tuberculosis, Mycobacterium avium,
Mycobacterium intracellulare, Mycobacterium kansaii, Mycobacterium
gordonae, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria
meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group
A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus viridans, Streptococcus faecalis, Streptococcus
bovis, Streptococcus pneumoniae, Haemophilus influenzae, Bacillus
antracis, Erysipelothrix rhusiopathiae, Clostridium tetani,
Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella
multocida, Fusobacterium nucleatum, Streptobacillus moniliformis,
Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia,
and Actinomyces israelii, Acinetobacter, Bacillus, Bordetella,
Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila,
Clostridium, Corynebacterium, Enterococcus, Haemophilus,
Helicobacter, Mycobacterium, Mycoplasma, Stenotrophomonas,
Treponema, Vibrio, Yersinia, Acinetobacter baumanii, Bordetella
pertussis, Brucella abortus, Brucella canis, Brucella melitensis,
Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae,
Chlamydia trachomatis, Chlamydophila psittaci, Clostridium
botulinum, Clostridium difficile, Clostridium perfringens,
Corynebacterium diphtheriae, Enterobacter sazakii, Enterobacter
agglomerans, Enterobacter cloacae, Enterococcus faecalis,
Enterococcus faecium, Escherichia coli, Francisella tularensis,
Helicobacter pylori, Legionella pneumophila, Leptospira
interrogans, Mycobacterium leprae, Mycobacterium tuberculosis,
Mycobacterium ulcerans, Mycoplasma pneumoniae, Pseudomonas
aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella
typhimurium, Salmonella enterica, Shigella sonnei, Staphylococcus
epidermidis, Staphylococcus saprophyticus, Stenotrophomonas
maltophilia, Vibrio cholerae, Yersinia pestis, and the like.
[0143] The biological sample may comprise one or more viruses.
Non-limiting examples of viruses include the herpes virus (e.g.,
human cytomegalomous virus (HCMV), herpes simplex virus 1 (HSV-1),
herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV),
Epstein-Barr virus), influenza A virus and Hepatitis C virus (HCV)
or a picornavirus such as Coxsackievirus B3 (CVB3). Other viruses
may include, but are not limited to, the hepatitis B virus, HIV,
poxvirus, hepadavirus, retrovirus, and RNA viruses such as
flavivirus, togavirus, coronavirus, Hepatitis D virus,
orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, filo virus,
Adenovirus, Human herpesvirus, type 8, Human papillomavirus, BK
virus, JC virus, Smallpox, Hepatitis B virus, Human bocavirus,
Parvovirus B19, Human astrovirus, Norwalk virus, coxsackievirus,
hepatitis A virus, poliovirus, rhinovirus, Severe acute respiratory
syndrome virus, Hepatitis C virus, yellow fever virus, dengue
virus, West Nile virus, Rubella virus, Hepatitis E virus, and Human
immunodeficiency virus (HIV). In some cases, the virus is an
enveloped virus. Examples include, but are not limited to, viruses
that are members of the hepadnavirus family, herpesvirus family,
iridovirus family, poxvirus family, flavivirus family, togavirus
family, retrovirus family, coronavirus family, filovirus family,
rhabdovirus family, bunyavirus family, orthomyxovirus family,
paramyxovirus family, and arenavirus family. Other examples
include, but are not limited to, Hepadnavirus hepatitis B virus
(HBV), woodchuck hepatitis virus, ground squirrel (Hepadnaviridae)
hepatitis virus, duck hepatitis B virus, heron hepatitis B virus,
Herpesvirus herpes simplex virus (HSV) types 1 and 2,
varicella-zoster virus, cytomegalovirus (CMV), human
cytomegalovirus (HCMV), mouse cytomegalovirus (MCMV), guinea pig
cytomegalovirus (GPCMV), Epstein-Barr virus (EBV), human herpes
virus 6 (HHV variants A and B), human herpes virus 7 (HHV-7), human
herpes virus 8 (HHV-8), Kaposi's sarcoma--associated herpes virus
(KSHV), B virus Poxvirus vaccinia virus, variola virus, smallpox
virus, monkeypox virus, cowpox virus, camelpox virus, ectromelia
virus, mousepox virus, rabbitpox viruses, raccoonpox viruses,
molluscum contagiosum virus, orf virus, milker's nodes virus, bovin
papullar stomatitis virus, sheeppox virus, goatpox virus, lumpy
skin disease virus, fowlpox virus, canarypox virus, pigeonpox
virus, sparrowpox virus, myxoma virus, hare fibroma virus, rabbit
fibroma virus, squirrel fibroma viruses, swinepox virus, tanapox
virus, Yabapox virus, Flavivirus dengue virus, hepatitis C virus
(HCV), GB hepatitis viruses (GBV-A, GBV-B and GBV-C), West Nile
virus, yellow fever virus, St. Louis encephalitis virus, Japanese
encephalitis virus, Powassan virus, tick-borne encephalitis virus,
Kyasanur Forest disease virus, Togavirus, Venezuelan equine
encephalitis (VEE) virus, chikungunya virus, Ross River virus,
Mayaro virus, Sindbis virus, rubella virus, Retrovirus human
immunodeficiency virus (HIV) types 1 and 2, human T cell leukemia
virus (HTLV) types 1, 2, and 5, mouse mammary tumor virus (MMTV),
Rous sarcoma virus (RSV), lentiviruses, Coronavirus, severe acute
respiratory syndrome (SARS) virus, Filovirus Ebola virus, Marburg
virus, Metapneumoviruses (MPV) such as human metapneumovirus
(HMPV), Rhabdovirus rabies virus, vesicular stomatitis virus,
Bunyavirus, Crimean-Congo hemorrhagic fever virus, Rift Valley
fever virus, La Crosse virus, Hantaan virus, Orthomyxovirus,
influenza virus (types A, B, and C), Paramyxovirus, parainfluenza
virus (PIV types 1, 2 and 3), respiratory syncytial virus (types A
and B), measles virus, mumps virus, Arenavirus, lymphocytic
choriomeningitis virus, Junin virus, Machupo virus, Guanarito
virus, Lassa virus, Ampari virus, Flexal virus, Ippy virus, Mobala
virus, Mopeia virus, Latino virus, Parana virus, Pichinde virus,
Punta toro virus (PTV), Tacaribe virus and Tamiami virus. In some
embodiments, the virus is a non-enveloped virus, examples of which
include, but are not limited to, viruses that are members of the
parvovirus family, circovirus family, polyoma virus family,
papillomavirus family, adenovirus family, iridovirus family,
reovirus family, birnavirus family, calicivirus family, and
picornavirus family. Specific examples include, but are not limited
to, canine parvovirus, parvovirus B19, porcine circovirus type 1
and 2, BFDV (Beak and Feather Disease virus, chicken anaemia virus,
Polyomavirus, simian virus 40 (SV40), JC virus, BK virus,
Budgerigar fledgling disease virus, human papillomavirus, bovine
papillomavirus (BPV) type 1, cotton tail rabbit papillomavirus,
human adenovirus (HAdV-A, HAdV-B, HAdV-C, HAdV-D, HAdV-E, and
HAdV-F), fowl adenovirus A, bovine adenovirus D, frog adenovirus,
Reovirus, human orbivirus, human coltivirus, mammalian
orthoreovirus, bluetongue virus, rotavirus A, rotaviruses (groups B
to G), Colorado tick fever virus, aquareovirus A, cypovirus 1, Fiji
disease virus, rice dwarf virus, rice ragged stunt virus,
idnoreovirus 1, mycoreovirus 1, Birnavirus, bursal disease virus,
pancreatic necrosis virus, Calicivirus, swine vesicular exanthema
virus, rabbit hemorrhagic disease virus, Norwalk virus, Sapporo
virus, Picornavirus, human polioviruses (1-3), human
coxsackieviruses Al-22, 24 (CAl-22 and CA24, CA23 (echovirus 9)),
human coxsackieviruses (Bl-6 (CB1-6)), human echoviruses 1-7, 9,
11-27, 29-33, vilyuish virus, simian enteroviruses 1-18 (SEV1-18),
porcine enteroviruses 1-11 (PEVl-11), bovine enteroviruses 1-2
(BEV1-2), hepatitis A virus, rhinoviruses, hepatoviruses, cardio
viruses, aphthoviruses and echoviruses. The virus may be phage.
Examples of phages include, but are not limited to T4, T5, .lamda.
phage, T7 phage, G4, P1, p.sup.6, Thermoproteus tenax virus 1, M13,
MS2, QP, gpX174, 129, PZA, D15, BS32, B103, M2Y (M2), Nf, GA-1,
FWLBc1, FWLBc2, FWLLm3, B4. In some cases, the virus is selected
from a member of the Flaviviridae family (e.g., a member of the
Flavivirus, Pestivirus, and Hepacivirus genera), which includes the
hepatitis C virus, Yellow fever virus; Tick-borne viruses, such as
the Gadgets Gully virus, Kadam virus, Kyasanur Forest disease
virus, Langat virus, Omsk hemorrhagic fever virus, Powassan virus,
Royal Farm virus, Karshi virus, tick-borne encephalitis virus,
Neudoerfl virus, Sofjin virus, Louping ill virus and the Negishi
virus; seabird tick-borne viruses, such as the Meaban virus,
Saumarez Reef virus, and the Tyuleniy virus; mosquito-borne
viruses, such as the Aroa virus, dengue virus, Kedougou virus,
Cacipacore virus, Koutango virus, Japanese encephalitis virus,
Murray Valley encephalitis virus, St. Louis encephalitis virus,
Usutu virus, West Nile virus, Yaounde virus, Kokobera virus, Bagaza
virus, Ilheus virus, Israel turkey meningoencephalo-myelitis virus,
Ntaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui virus,
Edge Hill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S
virus, Wesselsbron virus, yellow fever virus; and viruses with no
known arthropod vector, such as the Entebbe bat virus, Yokose
virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus,
Sal Vieja virus, San Perlita virus, Bukalasa bat virus, Carey
Island virus, Dakar bat virus, Montana myotis leukoencephalitis
virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, and
the Cell fusing agent virus. In some cases, the virus is selected
from a member of the Arenaviridae family, which includes the Ippy
virus, Lassa virus (e.g., the Josiah, LP, or GA391 strain),
lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia
virus, Amapari virus, Flexal virus, Guanarito virus, Junin virus,
Latino virus, Machupo virus, Oliveros virus, Parana virus, Pichinde
virus, Pirital virus, Sabia virus, Tacaribe virus, Tamiami virus,
Whitewater Arroyo virus, Chapare virus, and Lujo virus. In some
cases, the virus is selected from a member of the Bunyaviridae
family (e.g., a member of the Hantavirus, Nairovirus,
Orthobunyavirus, and Phlebovirus genera), which includes the
Hantaan virus, Sin Nombre virus, Dugbe virus, Bunyamwera virus,
Rift Valley fever virus, La Crosse virus, Punta Toro virus (PTV),
California encephalitis virus, and Crimean-Congo hemorrhagic fever
(CCHF) virus. In some cases, the virus is selected from a member of
the Filoviridae family, which includes the Ebola virus (e.g., the
Zaire, Sudan, Ivory Coast, Reston, and Uganda strains) and the
Marburg virus (e.g., the Angola, Ci67, Musoke, Popp, Ravn and Lake
Victoria strains); a member of the Togaviridae family (e.g., a
member of the Alphavirus genus), which includes the Venezuelan
equine encephalitis virus (VEE), Eastern equine encephalitis virus
(EEE), Western equine encephalitis virus (WEE), Sindbis virus,
rubella virus, Semliki Forest virus, Ross River virus, Barmah
Forest virus, O' nyong'nyong virus, and the chikungunya virus; a
member of the Poxyiridae family (e.g., a member of the
Orthopoxvirus genus), which includes the smallpox virus, monkeypox
virus, and vaccinia virus; a member of the Herpesviridae family,
which includes the herpes simplex virus (HSV; types 1, 2, and 6),
human herpes virus (e.g., types 7 and 8), cytomegalovirus (CMV),
Epstein-Barr virus (EBV), Varicella-Zoster virus, and Kaposi's
sarcoma associated-herpesvirus (KSHV); a member of the
Orthomyxoviridae family, which includes the influenza virus (A, B,
and C), such as the H5N1 avian influenza virus or H1N1 swine flu; a
member of the Coronaviridae family, which includes the severe acute
respiratory syndrome (SARS) virus; a member of the Rhabdoviridae
family, which includes the rabies virus and vesicular stomatitis
virus (VSV); a member of the Paramyxoviridae family, which includes
the human respiratory syncytial virus (RSV), Newcastle disease
virus, hendravirus, nipahvirus, measles virus, rinderpest virus,
canine distemper virus, Sendai virus, human parainfluenza virus
(e.g., 1, 2, 3, and 4), rhinovirus, and mumps virus; a member of
the Picornaviridae family, which includes the poliovirus, human
enterovirus (A, B, C, and D), hepatitis A virus, and the
coxsackievirus; a member of the Hepadnaviridae family, which
includes the hepatitis B virus; a member of the Papillamoviridae
family, which includes the human papilloma virus; a member of the
Parvoviridae family, which includes the adeno-associated virus; a
member of the Astroviridae family, which includes the astrovirus; a
member of the Polyomaviridae family, which includes the JC virus,
BK virus, and SV40 virus; a member of the Calciviridae family,
which includes the Norwalk virus; a member of the Reoviridae
family, which includes the rotavirus; and a member of the
Retroviridae family, which includes the human immunodeficiency
virus (HIV; e.g., types 1 and 2), and human T-lymphotropic virus
Types I and II (HTLV-1 and HTLV-2, respectively).
[0144] The biological sample may comprise one or more fungi.
Examples of infectious fungal agents include, without limitation
Aspergillus, Blastomyces, Coccidioides, Cryptococcus, Histoplasma,
Paracoccidioides, Sporothrix, and at least three genera of
Zygomycetes. The above fungi, as well as many other fungi, can
cause disease in pets and companion animals. The present teaching
is inclusive of substrates that contact animals directly or
indirectly. Examples of organisms that cause disease in animals
include Malasseziafurfur, Epidermophytonfloccosur, Trichophyton
mentagrophytes, Trichophyton rubrum, Trichophyton tonsurans,
Trichophyton equinum, Dermatophilus congolensis, Microsporum canis,
Microsporu audouinii, Microsporum gypseum, Malassezia ovale,
Pseudallescheria, Scopulariopsis, Scedosporium, and Candida
albicans. Further examples of fungal infectious agent include, but
are not limited to, Aspergillus, Blastomyces dermatitidis, Candida,
Coccidioides immitis, Cryptococcus neoformans, Histoplasma
capsulatum var. capsulatum, Paracoccidioides brasiliensis,
Sporothrix schenckii, Zygomycetes spp., Absidia corymbifera,
Rhizomucor pusillus, or Rhizopus arrhizus.
[0145] The biological sample may comprise one or more parasites.
Non-limiting examples of parasites include Plasmodium, Leishmania,
Babesia, Treponema, Borrelia, Trypanosoma, Toxoplasma gondii,
Plasmodium falciparum, P. vivax, P. ovale, P. malariae, Trypanosoma
spp., or Legionella spp. In some cases, the parasite is Trichomonas
vaginalis.
[0146] In some cases, the biological sample is a sample taken from
a subject infected with or suspected of being infected with an
infectious agent (e.g., bacteria, virus). In some aspects, the
biological sample comprises an infectious agent associated with a
sexually-transmitted disease (STD) or a sexually-transmitted
infection (STI). Non-limiting examples of STDs or STIs and
associated infectious agents that may be detected with the devices
and methods provided herein may include, Bacterial Vaginosis;
Chlamydia (Chlamydia trachomatis); Genital herpes (herpes virus);
Gonorrhea (Neisseria gonorrhoeae); Hepatitis B (Hepatitis B virus);
Hepatitis C (Hepatitis C virus); Genital Warts, Anal Warts,
Cervical Cancer (Human Papillomavirus); Lymphogranuloma venereum
(Chlamydia trachomatis); Syphilis (Treponema pallidum);
Trichomoniasis (Trichomonas vaginalis); Yeast infection (Candida);
and Acquired Immunodeficiency Syndrome (Human Immunodeficiency
Virus).
[0147] In some cases, the sample can be from an environmental
source or an industrial source. Examples of environmental sources
include, but are not limited to, agricultural fields, lakes,
rivers, water reservoirs, air vents, walls, roofs, soil samples,
plants, and swimming pools. Examples of industrial sources include,
but are not limited to clean rooms, hospitals, food processing
areas, food production areas, food stuffs, medical laboratories,
pharmacies, and pharmaceutical compounding centers. The sample can
be a forensic sample (e.g., hair, blood, semen, saliva, etc.). The
sample can comprise an agent used in a bioterrorist attack (e.g.,
influenza, anthrax, smallpox).
[0148] In some cases, more than one sample can be obtained from a
subject or source, and multiple immunoassay tests using a single
sensing device or apparatus described herein can be performed. In
some cases, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,
50 or more samples can be obtained. In some cases, more than one
sample may be obtained over a period of time, for example, to
monitor disease progression or to monitor a biological state or
condition (e.g., cardiac conditions). Generally, the sensing
devices of the disclosure are configured for repeated or continuous
use. Alternatively, the sensing devices can be one-time use (e.g.,
disposable).
[0149] In some cases, the subject is affected by a genetic disease,
a carrier for a genetic disease or at risk for developing or
passing down a genetic disease, where a genetic disease is any
disease that can be linked to a genetic variation such as
mutations, insertions, additions, deletions, translocation, point
mutation, trinucleotide repeat disorders and/or single nucleotide
polymorphisms (SNPs).
[0150] The biological sample can be from a subject who has a
specific disease, disorder, or condition, or is suspected of having
(or at risk of having) a specific disease, disorder or condition.
For example, the biological sample can be from a cancer patient, a
patient suspected of having cancer, or a patient at risk of having
cancer. The cancer can be, e.g., acute lymphoblastic leukemia
(ALL), acute myeloid leukemia (AML), adrenocortical carcinoma,
Kaposi Sarcoma, anal cancer, basal cell carcinoma, bile duct
cancer, bladder cancer, bone cancer, osteosarcoma, malignant
fibrous histiocytoma, brain stem glioma, brain cancer,
craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma,
medulloeptithelioma, pineal parenchymal tumor, breast cancer,
bronchial tumor, Burkitt lymphoma, Non-Hodgkin lymphoma, carcinoid
tumor, cervical cancer, chordoma, chronic lymphocytic leukemia
(CLL), chronic myelogenous leukemia (CML), colon cancer, colorectal
cancer, cutaneous T-cell lymphoma, ductal carcinoma in situ,
endometrial cancer, esophageal cancer, Ewing Sarcoma, eye cancer,
intraocular melanoma, retinoblastoma, fibrous histiocytoma,
gallbladder cancer, gastric cancer, glioma, hairy cell leukemia,
head and neck cancer, heart cancer, hepatocellular (liver) cancer,
Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal
cancer, lip cancer, oral cavity cancer, lung cancer, non-small cell
carcinoma, small cell carcinoma, melanoma, mouth cancer,
myelodysplastic syndromes, multiple myeloma, medulloblastoma, nasal
cavity cancer, paranasal sinus cancer, neuroblastoma,
nasopharyngeal cancer, oral cancer, oropharyngeal cancer,
osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis,
paraganglioma, parathyroid cancer, penile cancer, pharyngeal
cancer, pituitary tumor, plasma cell neoplasm, prostate cancer,
rectal cancer, renal cell cancer, rhabdomyosarcoma, salivary gland
cancer, Sezary syndrome, skin cancer, nonmelanoma, small intestine
cancer, soft tissue sarcoma, squamous cell carcinoma, testicular
cancer, throat cancer, thymoma, thyroid cancer, urethral cancer,
uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer,
Waldenstrom Macroglobulinemia, or Wilms Tumor. The sample can be
from the cancer and/or normal tissue from the cancer patient. In
some cases, the sample is a biopsy of a tumor.
[0151] The biological sample can be processed to render it
competent for performing any of the methods using any of the
devices or kits provided herein. For example, a solid sample may be
dissolved in a liquid medium or otherwise prepared as a liquid
sample to facilitate flow along the test strip of the device. In
such cases where biological cells or particles are used, the
biological cells or particles may be lysed or otherwise disrupted
such that the contents of the cells or particles are released into
a liquid medium. Molecules contained in cell membranes and/or cell
walls may also be released into the liquid medium in such cases. A
liquid medium may include water, saline, cell-culture medium, or
any solution and may contain any number of salts, surfactants,
buffers, reducing agents, denaturants, preservatives, and the
like.
[0152] Generally, the sample contains or is suspected of containing
one or more target analytes. In various aspects, the sample may
contain at least a first analyte and a second analyte. The term
"analyte" as used herein may refer to any substance that is to be
analyzed using the methods and devices provided herein. The
immunoassay sensing devices and arrays disclosed herein may be
configured to simultaneously detect the presence of 1, 2, 3, 4, 5,
6, 7, 8, 9, 10 or more analytes in a sample. The immunoassay
sensing devices and arrays disclosed herein can be capable of
simultaneous and multiplexed detection of multiple target analytes
in a single sample.
[0153] Non-limiting examples of analytes may include proteins,
haptens, immunoglobulins, hormones, polynucleotides, steroids,
drugs, infectious disease agents (e.g., of bacterial or viral
origin), drugs of abuse, environmental agents, biological markers,
and the like. In one case, the immunoassay detects at least a first
analyte, wherein the first analyte is luteinizing hormone (LH). In
another case, the immunoassay detects at least a first analyte,
wherein the first analyte is human chorionic gonadotropin (hCG). In
another case, the immunoassay detects at least a first analyte and
a second analyte, wherein the first analyte is
estrone-3-glucoronide (E3G) and the second analyte is luteinizing
hormone (LH). In another case, the immunoassay detects at least a
first analyte and a second analyte, wherein the first analyte is a
surface antigen on a first viral particle (e.g., Influenza A) and
the second analyte is a surface antigen on a second viral particle
(e.g., Influenza B). In another case, the immunoassay detects at
least a first analyte, wherein the first analyte is
25-hydroxyvitamin D, 25-hydroxyvitamin D2 [25(OH)D.sub.2], or
25-hydroxyvitamin D3 [25(OH)D.sub.3]. In another case, the
immunoassay detects at least a first analyte and a second analyte,
wherein the first analyte is triiodothyronine (T3) and the second
analyte is thyroxine (T4). In another case, the immunoassay detects
at least a first analyte, wherein the first analyte is an allergen.
Non-limiting examples of allergens may include: Balsam of Peru,
fruit, rice, garlic, oats, meat, milk, peanuts, fish, shellfish,
soy, tree nuts, wheat, hot peppers, gluten, eggs, tartrazine,
sulfites, tetracycline, phenytoin, carbamazepine, penicillin,
cephalosporins, sulfonamides, non-steroidal anti-inflammatories
(e.g., cromolyn sodium, nedocromil sodium, etc.), intravenous
contrast dye, local anesthetics, pollen, cat allergens, dog
allergens, insect stings, mold, perfume, cosmetics, semen, latex,
water, house dust mites, nickel, gold, chromium, cobalt chloride,
formaldehyde, photographic developers, fungicide,
dimethylaminopropylamine, paraphenylenediamine, glyceryl
monothioglycolate, toluenesulfonomide formaldehyde.
[0154] The sensing device may be used to test for the presence or
absence of at least a first analyte and a second analyte in a
sample. In some cases, the sensing device may be used to determine
an amount or a relative amount of at least a first and second
analyte in a sample.
[0155] The presence or absence of analytes may be indicative of a
disease or disorder in a subject. The presence or absence of
analytes may be indicative of a biological state or condition of a
subject. In some cases, the presence or absence of analytes
indicates that a subject has or is at risk of developing a disease.
In some cases, the presence or absence of analytes indicates that a
subject has a disorder (e.g., thyroid disorder). In some cases, the
presence or absence of analytes indicates that a subject has a
deficiency (e.g., vitamin deficiency). In some cases, the presence
or absence of analytes indicates that a product (e.g., a food or
drink product) contains an allergen.
[0156] G. Sensing Mechanisms
[0157] The sensing device 100 may be an electrochemical sensing
device configured for both catalytic and affinity-based detection
of one or more target analytes in a sample. A catalytic sensor(s)
or catalytic sensing utilizes molecules (such as enzymes) that
catalyze a biochemical reaction on the sensing surface with the
target molecule and detection based on the resulting products. An
affinity-based sensor(s) or affinity-based sensing is designed to
monitor binding of the target molecule and uses other specific
binding molecules (e.g., proteins, lectins, receptors, nucleic
acids, whole cells, aptamers, DNA/RNA, antibodies or
antibody-related substances, etc.) for biomolecular
recognition.
[0158] In many embodiments, the sensing devices or arrays disclosed
herein can be configured to simultaneously detect and quantitate
different isoforms of a single protein. The molecules associated
with the catalysis-based reaction may be anchored onto the sensing
surface (e.g. working electrode) through an affinity-based
mechanism to ensure that the chemical reaction(s) occurs in
proximity of the sensing surface for enhanced sensitivity of
detection. The output electrical signals for both catalytic and
affinity sensors/sensing is measured in current, voltage, and
impedance.
[0159] Amperometric (i.e. DC current--DC voltage--time) and
impedimetric sensors are electroanalytical methods for
characterization of the surface phenomena and changes at the
sensing electrode surfaces. Amperometric sensors can measure
changes to electric current resulting from either catalytic
mechanisms and/or affinity binding mechanisms occurring at the
sensing electrode surfaces under an applied field/potential and
that are related to the concentration of the target species or
analytes present in the solution. Voltammetry and chronoamperometry
are subclasses of amperometry. In voltammetry, current is measured
by varying the potential applied to the sensing electrode. In
chronoamperometry, current is measured at a fixed potential, at
different times after the start of sensing.
[0160] The aforementioned sensors and sensing methods are
particularly well-suited for detection of catalytic processes and
their associated effects modulated due to kinetic and thermodynamic
properties. Signal transduction and quantification occurs through
the dynamic transfer of electrons resulting from the catalytic
processes and/or the associated chemical reactions to the sensing
electrode surface. Specificity in detection of target species or
analytes can be achieved through the choice of the catalytic
processes and the higher reaction rate kinetics occurring within
the electrochemical potential window, which can result in amplified
signals through the sensing electrode surface.
[0161] Impedimetric sensors are well-suited for detection of
binding events on the sensing electrode surface. Analytes can
interact with the sensing electrode through selective treatments
applied to the electrode surface in the form of cross-linkers
(e.g., antibodies, nucleic acids, ligands, etc.) that are
covalently conjugated onto sensing electrode surface. The impedance
Z of the sensor can be determined by applying a voltage
perturbation with a small amplitude and detecting the current
response. The impedance Z is the quotient of the voltage-time
function V(t) and the resulting current-time function I(t), and
given as follows:
Z = V ( t ) I ( t ) = V 0 sin ( 2 .PI. f t ) I 0 sin ( 2 .PI. f t +
.phi. ) = 1 Y ##EQU00001##
where V.sub.0 and I.sub.0 are the maximum voltage and current
signals, f is the frequency, t the time, .phi. the phase shift
between the voltage-time and current-time functions, and Y is the
complex conductance or admittance. The measured impedance
associated with biomolecule binding is a complex value, since the
current can differ in terms of not only the amplitude but also it
can show a phase shift .phi. compared to the voltage-time function.
Thus, the value can be described either by the modulus |Z| and the
phase shift .phi. or alternatively by the real part ZR and the
imaginary part ZI of the impedance. Therefore, the results of an
impedance measurement can be illustrated in two different ways:
using a Bode plot, which plots log |Z| and .phi. as a function of
log f, or using a Nyquist plot, which plots ZR and ZI. Both of
these plots can be used to establish calibration responses of the
sensing device towards real-time detection and quantification of
the target species or analytes. Sensitivity and specificity in
detection can be achieved through deconstruction of the Nyquist and
Bode plots, by identifying the frequency range where the electrical
double layer effects due to the binding events of the target
species occur and quantifying the change in impedance with
concentration within this range.
[0162] In various embodiments, when a working electrode comprising
ZnO nanostructures is exposed to a sample (e.g., an ionic solution
comprising biomolecules), a potential difference is generated at
the electrode/electrolyte interface due to the unequal distribution
of charges. As a consequence of biomolecular binding events at the
surface of the ZnO nanostructures, redistribution of charges in the
working electrode and ions in the electrolyte can result in
formation of a space-charge region within the ZnO nanostructures
and an electrical double layer at the interface between the
electrode and the electrolyte. Evaluation and quantification of
biomarker binding can be achieved by measuring the changes in
electrode resistance or capacitance at selected frequencies.
[0163] The changes to space-charge capacitance and overall
impedance at the ZnO nanostructures/electrolyte interfaces can be
characterized by respectively using a direct current (DC)-based
Mott-Schottky technique and an alternating current (AC)-based
electrochemical impedance spectroscopy (EIS) technique towards
detection of target analytes or biomarkers. Correlation in output
signal response with concentration can be established between the
DC and AC electrochemical detection techniques.
[0164] As previously described, the plurality of capture reagents
of the sensing device are configured to selectively bind to one or
more target analytes in a sample, thereby effecting changes to
electron and ion mobility and charge accumulation in different
regions of the semiconducting nanostructures and the sample. The
changes to the electron and ion mobility and charge accumulation
can be detected with aid of sensing circuitry, and can be used to
determine a presence and concentration of the one or more target
analytes in the sample. The changes to the electron and ion
mobility and charge accumulation can be transduced into electrical
impedance and capacitance signals. The signals may be indicative of
interfacial charge modulation comprising of the changes to the
electron and ion mobility. Additionally, the signals may be
indicative of capacitance changes to a space-charge region formed
in the semiconducting nanostructures upon binding of the one or
more target analytes to the capture reagents. The changes may
comprise simultaneous modulation to the ion mobility in one or more
regions adjacent or proximal to the semiconducting
nanostructures.
[0165] The sensing circuitry may comprise hardware, software, or a
combination of software and hardware. The sensing circuitry may
comprise a single or multiple microprocessors, field programmable
gate arrays (FPGAs), or digital signal processors (DSPs). The
sensing circuitry may be electrically connected to the sensing
device. In some embodiments, the sensing circuitry may be part of
the sensing device, for example the sensing circuitry may be
assembled or disposed on the substrate. Alternatively, the sensing
circuitry may be remote to the sensing device.
[0166] The sensing circuitry can be configured to implement a
plurality of electrochemical detection techniques for detecting the
capacitance changes and impedance changes. The plurality of
electrochemical detection techniques may comprise, for example (1)
a modified Electrochemical Impedance Spectroscopy (EIS) technique
for measuring the impedance changes and (2) Mott-Schottky technique
for measuring the capacitance changes. The modified EIS technique
is capable of distinguishing the electrical impedance signals from
background noise at low concentrations of the target analytes in
the sample. The sensing circuitry can be configured to analyze the
electrical impedance and capacitance signals by concurrently
analyzing a set of Nyquist plots obtained via the modified EIS
technique and a set of Mott-Schottky plots obtained via the
Mott-Schottky technique. The modified EIS technique may comprise
(1) sectioning an interfacial charge layer into a plurality of
spatial dielectric z-planes along a direction orthogonal to the
interface between the fluid sample and the semiconducting
nanostructures, and (2) probing each of the plurality of z-planes
with a specific frequency selected from a range of frequencies.
Specific binding of different target analytes to the capture
reagents may occur at known spatial heights within the interfacial
charge layer. Accordingly, the sensing circuitry can be configured
to determine the presence and concentration of each of the
different target analytes by measuring the capacitance and
impedance changes at specific frequencies corresponding to their
respective z-planes at the known spatial heights within the
interfacial charge layer.
[0167] H. Room-Temperature Ionic Liquids (RTIL)
[0168] The inherent non-stoichiometric nature of ZnO may result in
generation of oxygen vacancies, and the ease in forming surface
bonds with hydroxyl molecules and other ions can render the ZnO
surface sensitive to the pH of the biofluids and environment. Thus,
ZnO-based sensing devices may develop drifts in signal output over
time, independent of detection modality, especially when exposed to
varying pH solutions in the presence of enzymatic reactions that
involve generation of hydrogen peroxide. In addition, protein
biomolecules can easily denature when exposed to temperature,
environment, and pH outside the established range of their
stability.
[0169] To mitigate the above effects, a sample may be provided in a
room temperature ionic liquid (RTIL) electrolyte buffer in some
embodiments. The stability and reliability of the bound proteins to
the functionalized nanostructured ZnO surfaces can be improved with
the use of RTIL as the electrolyte solvent buffer containing the
specific protein antibodies, and that can conjugate with the
functionalized ZnO surface during the immunoassay steps. The RTIL
can also provide stability of the bound proteins during subsequent
storage and handling and from exposure to environment. In simple
electrolyte solvent solutions, the protein charge is typically
determined by the equilibrium protonation of hydroxyl- and
amino-groups, and depends on the pH of the environment, whose
variations can even reverse the sign of the overall charge. In
contrast, for RTILs, dispersion energy, ion size, and additional
H-bonding sites can be useful in determining protein
characteristics. Unlike molecular solvents that are charge neutral,
RTILs are molten salts at room temperature composed solely of
polyatomic cations and anions.
[0170] The properties of RTILs can be changed according to the
requirement by modifying their constituents (cation and anion).
Although they can stabilize the protein over a wide range of
temperature, the thermal stability of proteins depends on the
appropriate choice of RTILs as proteins are not homogeneously
stable in all type of RTILs. In some cases, the stability and
activity of proteins is affected by many factors such as polarity,
hydrophilicity vs. hydrophobicity and hydrogen-bond capacity of
RTILs, excipients, and impurities. RTILs containing chaotropic
(large-sized and low charged, weakly hydrated ions that decrease
the structure of water) cations and kosmotropic (small-sized and
high charged, strongly hydrated ions that increase the structure of
water) anions can optimally stabilize the biological
macromolecules. In some embodiments, the kosmotropicity order of
anions and cations can be determined by using viscosity
B-coefficients and other parameters such as hydration entropies,
hydration volumes, heat capacity, NMR B-coefficients and ion
mobility.
[0171] In one embodiment, RTILs containing chaotropic cations and
kosmotropic anions can be selected to independently and optimally
stabilize the target proteins chosen i.e. cTnI and/or cTnT,
NT-proBNP, and CRP. Intermixing of protein biomolecules and
ensuring cross-reactivity response is well below the noise
threshold in signal transduction response from each of the bound
antibodies in the detection of their specific target proteins can
be achieved.
[0172] III. Multi-Configurable Sensing Array
[0173] In some embodiments, the plurality of semiconducting
nanostructures may be disposed on two or more electrodes comprising
of a first electrode and a second electrode. A first capture
reagent may be attached to the semiconducting nanostructures on the
first electrode and configured to selectively bind to a first
target analyte. A second capture reagent may be attached to the
semiconducting nanostructures on the second electrode and
configured to selectively bind to a second target analyte. The
sensing device is capable of simultaneously determining the
presence and concentrations of the first and second target analytes
upon binding of the target analytes to the respective capture
reagents.
[0174] In some embodiments, the first electrode may be part of a
first sensing device, and the second electrode may be part of a
second sensing device. The first and second sensing devices may be
provided on a common sensing platform. For example, FIG. 2 shows a
sensing array 200 comprising a plurality of sensing devices 100 for
detecting a plurality of different target analytes in a fluid
sample. The array may comprise two or more sensing devices (e.g.,
100-1 through 100-n, where n can be any integer greater than two)
disposed on a common substrate 210. Alternatively, the sensing
devices may be provided separately and then assembled onto the
substrate 210. The sensing devices may each comprise a working
electrode having a plurality of semiconducting nanostructures
disposed thereon and a capture reagent attached to the
semiconducting nanostructures. The sensing devices may or may not
have the same type of semiconducting nanostructures or materials.
The sensing devices may comprise different capture reagents that
are configured to selectively bind to the different target analytes
in the fluid sample. The selective binding is configured to effect
changes to electron and ion mobility and charge accumulation in
different regions of the semiconducting nanostructures and the
fluid sample. Each of the sensing devices can be configured to
determine a presence and concentration of a different target
analyte in the fluid sample based on detected changes to the
electron and ion mobility and charge accumulation.
[0175] A method of detecting a plurality of different target
analytes in a fluid sample may include providing the sensing array
described herein, and applying the fluid sample containing one or
more target analytes to the sensing array. The method may include
using each of the sensing devices to determine the presence and
concentration of a different target analyte in the fluid sample,
based on the detected changes to the electron and ion mobility and
charge accumulation in the different regions of the semiconducting
nanostructures and the fluid sample.
[0176] In some embodiments, an array 200 may comprise a first
sensing device 100-1 and a second sensing device 100-2 capable of
simultaneously determining the presence and concentrations of first
and second target analytes upon binding of the target analytes to
the respective capture reagents. In some embodiments, the first and
second target analytes may comprise different isoforms of a same
type of biomarker. In some embodiments, the target analytes may
comprise a plurality of cardiac biomarkers, and the plurality of
capture reagents may comprise a plurality of antibodies that are
specific to the plurality of cardiac biomarkers.
[0177] A. Simultaneous and Multiplexed Detection of Multiple Target
Analytes
[0178] As noted previously, there is a need for the rapid,
quantitative, specific, and multiplex detection and measurement of
target analyte concentrations at point of care. The ability to
perform multiplexed detection can provide significant advantages
for point of care diagnostics in that it allows for the
simultaneous monitoring of multiple markers in a single sample. The
multiplexing can support the performance of both negative and
positive controls in the same sample. Together, these attributes
can significantly improve the specificity and sensitivity with
which certain diseases and physiological conditions can be detected
and diagnosed.
[0179] The array 200 shown in FIG. 2 is capable of simultaneous and
multiplexed detection of different target analytes present in a
fluid sample using a plurality of electrochemical detection
techniques. FIG. 3 shows a multi-configurable sensing array 300
comprising a plurality of sensing devices 100-1, 100-2, 100-3
through 100-n. The electrodes of the sensing devices can be
connected to sensing circuitry configured for simultaneous
acquisition and multiplexing of electrical signals from the sensing
devices. The sensing devices can be configured for both catalytic
and affinity-based sensing. A working electrode in each sensing
device can be independently functionalized for specific detection
of a target analyte which may be a biomarker. Different sensing
devices in the array 300 may comprise different capture reagents
that are configured to selectively bind to the different target
analytes in the fluid sample. The output from each sensing device
may be independently measured and transduced (e.g., amperometric or
impedometric) to provide a combinatorial/multiplexed result
relating to the end physiological state being predicted. For
example, D.sub.12 may be the multiplexed result between sensing
devices 100-1 and 1002; D.sub.23 may be the multiplexed result
between sensing devices 100-2 and 1003; D.sub.13 may be the
multiplexed result between sensing devices 100-1 and 1003; Din may
be the multiplexed result between sensing devices 100-1 and 100-n,
and so forth. In some embodiments, the output from more than two
sensing devices, or all of the sensing devices, may be
independently measured and transduced (e.g., amperometric or
impedometric) to provide a combinatorial/multiplexed result
relating to the end physiological state being predicted. For
example, D.sub.123 . . . n may be the multiplexed result between
sensing devices 100-1, 100-2, 100-3 through 100-n. Any number or
combination of multiplexed results from the sensing devices may be
contemplated. The output from the two or more sensing devices can
be weighed the same (e.g. each output accorded a same weight) or
weighed differentially (e.g. different outputs accorded different
weights). In some embodiments, the output from a sensing device may
be compared or correlated with the output(s) of one or more other
sensing devices. For example, the output from sensing device 100-1
may be compared or correlated with the output(s) of one or more
other sensing devices (e.g, 100-2, 100-3) to improve specificity
and sensitivity in detecting and diagnosing certain diseases and
physiological conditions.
[0180] The multi-configurable array 300 can be configured for
detection of multiple analytes that may be useful in disease
detection. In some embodiments, the array can be used for paired
and simultaneous detection of disease markers in body fluids in a
non-invasive manner such as: (a) Inflammatory marker, interleukin-6
(IL-6) and diabetes marker, Glucose in human sweat; and/or (b)
Inflammatory markers, interleukin-6 (IL-6) and C-reactive protein
(CRP) and muscular dystrophy markers, creatine kinase (CK-MB) in
finger pricked capillary blood. In some embodiments, the array can
be integrated with other sensors within wearable fabric, devices,
and medical instruments such as strips, catheters, probes, patches
for non-communicable disease diagnosis such as cardiac, cancer,
Alzheimer's, muscular dystrophy, inflammatory markers, etc.
[0181] The array 300 may be capable of supporting simultaneous
detection of multiple target analytes in a single sample volume.
The volume may be 150 .mu.L, 140 .mu.L, 130 .mu.L, 120 .mu.L, 110
.mu.L, 100 .mu.L, 90 .mu.L, 80 .mu.L, 70 .mu.L, 60 .mu.L, 50 .mu.L,
40 .mu.L, 30 .mu.L, 20 .mu.L, 10 .mu.L, 1 .mu.L, or any value
therebetween. In some embodiments, the array 300 may be capable of
supporting simultaneous detection of multiple target analytes in a
single, submilliliter sample volume (e.g. <30 .mu.L). In some
embodiments, simultaneous and multiplexed detection of the target
analytes can be completed in a short time (e.g., on the order of a
few minutes or less), and using <20 .mu.L of sample volume. In
some embodiments, simultaneous and multiplexed detection of the
target analytes can be achieved using about 10-20 .mu.L of sample
volume.
[0182] B. Electrode Configurations
[0183] FIG. 4 shows an array 400 comprising a first sensing device
100-1 and a second device 100-2 in accordance with some
embodiments. The first and second sensing devices may be similar to
the sensing devices described elsewhere herein. In the example of
FIG. 4, the first and second sensing devices may share a common
reference electrode (RE) 130, instead of each sensing device having
its own reference electrode. The common reference electrode can
provide a stable and known electrode potential to the
electrochemical cell comprising of the first and second sensing
devices. The first and second sensing devices can operate based on
the same reference electrode potential, thereby permitting
simultaneous and multiplexed detection of target analytes, and
calibration of results between the two sensing devices.
[0184] The first sensing device 100-1 may comprise a working
electrode (WE) 120-1 and a counter electrode (CE) 140-1. The second
sensing device 100-2 may comprise a working electrode (WE) 120-2
and a counter electrode (CE) 140-2. The common RE 130 may be
disposed between the working electrodes of the two sensing devices.
The common RE 130 may also be disposed between the counter
electrodes of the two sensing devices. The WE 120-1, RE 130, and CE
140-1 may be located in proximity to each other in a first region
of the substrate 210. The WE 120-2, RE 130, and CE 140-2 may be
located in proximity to each other in a second region of the
substrate 210. The first and second regions may be part of a test
zone 150. The first sensing device may comprise a first capture
reagent configured to selectively bind to a first target analyte.
The second sensing device may comprise a second capture reagent
configured to selectively bind to a second target analyte. In some
embodiments, the common RE 130 may have a larger surface area than
each of the working electrodes and counter electrodes. For example,
the surface areas of WE:CE:RE may be designed in the ratio of 1:1:4
to ensure sufficient output signal response due to binding events
at the working electrodes.
[0185] IV. Sensing System
[0186] FIG. 5 shows a sensing system 500 in accordance with some
embodiments. The system 500 may comprise a multi-configurable array
of sensing devices, for example array 400 described with reference
to FIG. 4. The array 400 may comprise a first sensing device and a
second sensing device as described elsewhere herein. The first
sensing device may include a first working electrode (WE) 120-1 and
a first counter electrode (CE) 140-1. The second sensing device may
include a second working electrode (WE) 120-2 and a second counter
electrode (CE) 140-1. The first and second sensing devices may
share a common reference electrode (RE) 130.
[0187] FIG. 5 further shows a magnified schematic view of the
functionalized working electrode (WE) 120 of each sensing device.
As previously described, each working electrode can be
independently functionalized for specific detection of a target
biomarker(s). The output from each sensing device can be
independently measured and transduced (e.g., amperometric or
impedometric) to provide a multiplexed outcome relating to the end
physiological state being predicted.
[0188] Referring to FIG. 5, a plurality of semiconducting
nanostructures 122 may be disposed on the WEs 120. For example,
first semiconducting nanostructures 122-1 may be disposed on the
surface of the first WE 120-1, and second semiconducting
nanostructures 122-2 may be disposed on the surface of the second
WE 120-2. In some embodiments, the first and second semiconducting
nanostructures may be formed of a same semiconductor or
semiconductor alloy material. Alternatively, the first and second
semiconducting nanostructures may be formed of different types of
semiconductor or semiconductor alloy material. In some instances,
each of the first and second semiconducting nanostructures may
comprise two or more types of semiconductor or semiconductor alloy
material. The semiconducting nanostructures can be grown or
deposited on the surface of the working electrodes. In some
embodiments, the first and second semiconducting nanostructures may
comprise ZnO nanostructures, as described in more detail with
reference to FIGS. 6A-C.
[0189] FIG. 6A shows an SEM micrograph of ZnO nanostructures that
are selectively grown on the working electrodes of the sensing
array using low temperature aqueous hydrothermal growth mechanism.
The nanostructures may be elongated, and may include nanorods or
nanopillars. In some embodiments, the nanostructures may have an
aspect ratio of about 1:4. The nanostructures may be formed having
different shapes, sizes, dimensions, and/or aspect ratios depending
on the growth conditions. In some embodiments, the ZnO
nanostructures may be grown by tuning the chemical reactions
between the precursors Zn(NO3)2.6H2O and HMTA dissolved in water.
The thermal decomposition and hydrolysis reactions of these
precursors results in the formation of zinc hydroxyl species which
upon dehydration form ZnO nuclei. Pre-seeded regions on the working
electrodes can then act as nucleation sites for the aligned growth
of ZnO nanostructures. The higher surface energy difference between
polar and non-polar planes derives faster growth of ZnO along polar
planes resulting in c-axis oriented crystalline growth of wurtzite
ZnO nanostructures. The SEM micrograph in FIG. 6A shows the
morphology of synthesized ZnO nanostructures as vertically grown
hexagonal shaped rod-like structures and uniform growth on the
working electrodes. The SEM characterization indicates uniform
growth of hexagonal shaped ZnO nanostructures at the pre-seeded
working electrodes. The as-synthesized ZnO nanostructures can be
used to aid detection of various target analytes (e.g. cardiac
biomarkers) using the sensing array of FIGS. 4 and 5 as described
elsewhere herein.
[0190] FIG. 6B is an ATR-FTIR spectra showing evidence of DSP
functionalization on nanostructured ZnO sensing surface in the
range between 2000 cm.sup.-1 and 500 cm.sup.-1. FIG. 6C is an
ATR-FTIR spectra showing evidence of antibody immobilization on
nanostructured ZnO sensing surface in the range between 2000
cm.sup.-1 and 500 cm.sup.-1. Referring to FIG. 6B,
functionalization of ZnO nanostructures with linking reagent (e.g.
thiol-based DSP linker molecules) can provide binding sites for
immobilization of the capture reagent (e.g. antibodies). The peak
at 571 cm.sup.-1 is associated with the ZnO nanostructures and is
stable as the immunoassay is being conducted on the sensing array.
The peaks observed at 1053 cm.sup.-1 and 1314 cm.sup.-1 are
assigned to stretching vibrations of v(C--O) and v(N--O)
respectively. The spectral features v(C--O) is characteristic of
the ester linkage and v(N--O) represents the symmetric stretch of
nitro groups both of which disappears with immobilization of the
antibody molecule. The other succinimidyl identifier groups that
show evidence of DSP binding to ZnO surfaces are the carbonyl
stretch in primary amides (v(C.dbd.O)) at 1662 cm.sup.-1 and
bending vibrations of alkane stretch (v(C--H)) with two peaks at
2915 cm.sup.-1 and 3000 cm.sup.-1 (not shown). Bands assigned at
1411 cm.sup.-1 and 1436 cm.sup.-1 are characteristic of methylene
scissors deformation in the bound DSP molecule. Referring to FIG.
6C, appearance of broad band between 1200 cm.sup.-1 and 1020
cm.sup.-1 in the spectra is characteristic of v(C--C, C--N) and
confirms aminolysis of NHS groups in DSP with primary amines in
antibody establishing a stable conjugation of the antibody to the
linker functionalized ZnO nanostructure surfaces grown on Au
working electrodes.
[0191] The ATR-FTIR spectras of the surface functionalized ZnO
nanostructures (shown in FIGS. 6B and 6C) can be obtained using an
FTIR spectrometer equipped with a deuterated, L-alanine doped
triglycine sulfate (DLaTGS) Detector with KBr window and validation
motor. The spectrometer can be fitted with a sampling stage
equipped with a 600 diamond ATR crystal and the sample can be held
with a swivel clamp that applied an even and constant force during
the acquisition of the spectra. Each FT-IR spectrum collected on
the sample represents the average of 200 scans at 4 cm.sup.-1
resolution in the scan range of 4000-400 cm.sup.-1.
[0192] The samples for FTIR analysis can be prepared as follows:
(1) deposit a thin layer of gold (dimensions) on the glass slides
followed by ZnO seed deposition; (2) clean the glass slides
subsequently in acetone, isopropyl alcohol and deionized water
prior to use; (3) grow the ZnO nanostructures on seeded substrates
and wash with DI water to remove growth residues; (4) treat the
nanostructured ZnO substrates with 10 mM DSP in DMSO for an hour;
(5) after DSP functionalization, rinse the substrates with DMSO to
remove unbound molecules and stored with silica desiccants for
analysis. Some of the samples are washed .alpha.-cTnI antibody.
After 30 minutes, the antibody treated substrates are washed with
PBS and the FTIR analysis is then performed.
[0193] Referring back to FIG. 5, a plurality of capture reagents
124 may be directly or indirectly attached to the plurality of
semiconducting nanostructures 122. In some embodiments, a sample
comprising the target analytes 128 may be provided with a blocking
buffer. The blocking buffer may comprise a protein 125 that can
block or cap the binding sites of excess linking reagents that did
not bind to a capture reagent. The blocking buffer can improve the
signal-to-noise ratio of the sensing device. As shown in FIG. 5, a
first capture reagent 124-1 may be attached to the first
semiconducting nanostructures 122-1 on the first electrode 120-1,
and configured to selectively bind to a first target analyte 128-1.
A second capture reagent 124-2 may be attached to the second
semiconducting nanostructures 122-2 on the second electrode 120-2,
and configured to selectively bind to a second target analyte
128-2. In some embodiments, the semiconducting nanostructures 122-1
and 122-2 may be functionalized with a linking reagent 126, and the
capture reagents 124-1 and 124-2 may be immobilized onto the
semiconducting nanostructures 122-1 and 122-2 via the linking
reagent 126, as described in more detail with reference to FIGS.
7A-C.
[0194] In some embodiments, a working electrode may preferably
include a Au surface which offers ease of functionalization with
organic linker molecules with thiol, carboxylic, etc. terminal
ends. The terminal ends of the organic linker molecules bind to the
Au surface through adsorption processes and are thermodynamically
stable. In some embodiments, the WE may have an immersion Au
surface finish which has energetically favored sites for binding of
the terminal ends of the organic linker molecules in comparison to
other types of thin film Au deposition methods (example:
evaporation, sputtering, etc.). In other embodiments, the WE may
have an immersion Ag surface, except the Ag surface tends to
oxidize more easily than Au surface. A sensing WE with
semiconducting ZnO, TiO.sub.2, or MoS.sub.2 layers can be
functionalized with selective linker chemistry that subsequently
conjugate with capture reagents (e.g. biomolecules, small organic
molecules, etc.) required for target analyte recognition. In some
embodiments, a sensing WE with semiconducting ZnO, TiO.sub.2, or
MoS.sub.2 layers can be functionalized with non-biological chemical
capture reagents, for example for the detection of certain
chemicals or chemical compounds in the sample.
[0195] The selection of linker molecules can be influenced by
several factors including bond-stability, position of functional
groups, pH, presence/absence of amine groups for interaction with
antibody, surface charge etc. The availability of different
functional groups in linker molecules can enable the immobilization
of antibody through stable covalent linkage, and the
antibody-antigen interactions provide specificity for detection of
target analytes. In the embodiments described herein, binding of
capture reagents and subsequent biomolecules to the affinity
immunoassay leads to changes in the ion diffusion profile near the
nanostructures and hence changes in electrical properties
(capacitance, resistance, etc.). The electrochemical detection
methods described herein include means to directly characterize the
capture reagent--target analyte interactions based on charge
perturbations at the electrode-electrolyte interface. In some
embodiments, functionalization may include the use of thiol and
phosphonic acid terminated groups on ZnO nanostructures or thin
films.
[0196] FIG. 7A shows the functionalization of a sensing WE using
the linker molecule dithiobis(succinimidyl propionate) (DSP) in
accordance with an embodiment. The DSP contains an amine-reactive
N-hydroxysuccinimide (NHS) ester at each end of an 8-carbon spacer
arm containing a cleavable disulfide bond. The DSP reacts with the
Au surface to form stable Au-thiol bonds from which the
amine-reactive NHS ester extend. The NHS esters react with primary
amines at pH 7-9 to form stable amide bonds, along with release of
the N-hydroxy-succinimide leaving group. Proteins, including
antibodies, generally have several primary amines in the side chain
of lysine (K) residues and the N-terminus of each polypeptide that
are available as targets for NHS-ester crosslinking reagents. FIG.
7B shows the functionalization of a sensing WE using phosphoric
based organic linker molecules in accordance with another
embodiment, that can form stable Au-phoshonic bonds represented by
bond configurations a-e. Capture reagents (e.g., biomolecules) can
include proteins, small molecules, antibodies, nucleic acids, etc.,
and can be customized for the binding and detection of specific
target analytes of interest. The process of immobilizing the
capture reagents on the functionalized sensing WE surfaces and the
subsequent detection of biomarkers may be described as an assay.
FIG. 7C shows a schematic reaction for amine-reactive NHS ester
reagents with primary amines on a protein at pH 7-9 to form stable
amide bonds, along with release of the N-hydroxy-succinimide
leaving group. Proteins, including antibodies, generally have
several primary amines in the side chain of lysine (K) residues and
the N-terminus of each polypeptide that are available as targets
for NHS-ester crosslinking reagents. FIG. 7D illustrates a DSP
functionalized sensing WE surface forming stable amide bonds with
the primary amine groups of a selected antibody of interest.
[0197] Accordingly, the multi-configurable sensing array described
herein may comprise sensing working electrodes that can be
independently functionalized with the appropriate linker chemistry
and different capture reagents that are specific to the detection
of different target analytes. Affinity-based sensors/sensing can
leverage the above functionalization strategies. In catalytic-based
sensors/sensing, binding of catalysts to the electrode surfaces can
ensure that the chemical reaction and electron transfer occur in
proximity to the electrode surfaces.
[0198] A. Multiplexer and Sensing Circuitry
[0199] Referring back to FIG. 5, the sensing system 500 may further
comprise a multiplexer 150, sensing circuitry 160, and computing
device 170. The array 400 may be electrically connected to the
multiplexer 150 and the sensing circuitry 160. The multiplexer may
comprise a plurality of channels 152 for multiplexing electrical
signals received from the array. The first sensing device 100-1 may
be connected to a first channel 152-1 and the second sensing device
100-2 may be connected to a second channel 152-2. Referring to FIG.
5, the first WE 120-1, CE 140-1, and RE 130 may be connected to the
first channel 152-1. The second WE 120-2, CE 140-2, and RE 130 may
be connected to the second channel 152-2. The multiplexer 150 may
be in two-way communication with the sensing circuitry 160. For
example, the sensing circuitry can be configured to apply
modulation signals to the array via the multiplexer. Output signals
from the first and second channels may be transmitted to the
sensing circuitry for simultaneous and multiplexed detection of the
different target analytes present in the fluid sample.
[0200] The sensing circuitry 160 can be configured to take
electrochemical measurements. In some embodiments, the sensing
circuitry may comprise a potentiostat. The sensing circuitry may be
capable of signal generation and signal conditioning. In some
embodiments, the sensing circuitry may include converters such as
analog-to-digital converters (ADC) and digital-to-analog converters
(DAC). The sensing circuitry 160 can be configured to selectively
apply a plurality of modulation signals to the two sensing devices
100-1 and 100-2 to enable detection of the plurality of different
target analytes in the fluid sample. The sensing circuitry can be
configured to individually and selectively control, activate, or
modulate the two sensing devices. The plurality of modulation
signals can be configured to aid in enhancing detection sensitivity
of the different target analytes. The sensing arrays described
herein can include any number of electrodes (e.g. working
electrodes, counter electrodes, and reference electrodes) in
various types of configurations. The sensing circuitry can be
configured to individually and selectively control, activate, or
modulate any number of sensing devices by applying different
signals to the electrodes, for example as shown by the electrical
field simulations in FIGS. 17A-17F.
[0201] As previously described, the first and second sensing
devices 100-1 and 100-2 may comprise different capture reagents
124-1 and 124-2 that are configured to selectively bind to
different target analytes 128-1 and 128-2 in a fluid sample. The
selective binding is configured to effect changes to electron and
ion mobility and charge accumulation in different regions of the
semiconducting nanostructures 122-1 and 122-2 and the fluid sample.
Each of the sensing devices can be configured to determine a
presence and concentration of a different target analyte in the
fluid sample based on detected changes to the electron and ion
mobility and charge accumulation.
[0202] The sensing circuity 160 can be configured for simultaneous
acquisition and multiplexing of electrical signals from the sensing
devices 100-1 and 100-2. The sensing circuitry is configured to
analyze the electrical signals comprising of impedance and
capacitance signals. The signals may be indicative of interfacial
charge modulation comprising of the changes to the electron and ion
mobility. The signals may include capacitance changes to
space-charge regions formed in the semiconducting nanostructures
upon binding of the different target analytes to the corresponding
capture reagents. The changes may comprise simultaneous modulation
to the ion mobility in one or more regions adjacent to the
semiconducting nanostructures.
[0203] The sensing circuitry 160 can be configured to implement a
plurality of electrochemical detection techniques for detecting the
impedance changes and the capacitance changes. In some embodiments,
the plurality of electrochemical detection techniques may comprise
a modified EIS technique for measuring the impedance changes and
Mott-Schottky technique for measuring the capacitance changes. The
modified EIS technique is capable of distinguishing the electrical
impedance signals from background noise at low concentrations of
the different target analytes in the fluid sample.
[0204] The array 400 is capable of simultaneous and multiplexed
detection of the different target analytes present in the fluid
sample using the plurality of electrochemical detection techniques
with aid of the sensing circuitry 160. The sensing circuitry 160
can be configured to perform the simultaneous and multiplexed
detection by analyzing the electrical impedance and capacitance
signals to determine the presence and concentration of each of the
different target analytes. The sensing circuitry can be configured
to perform the simultaneous and multiplexed detection substantially
in real-time upon binding of the different target analytes to the
corresponding capture reagents on the semiconducting
nanostructures. The sensing circuitry can be configured to analyze
the impedance and capacitance signals by concurrently analyzing a
set of Nyquist plots obtained via the modified EIS technique and a
set of Mott-Schottky plots obtained via the Mott-Schottky
technique.
[0205] In some embodiments, the modified EIS technique may comprise
(1) sectioning an interfacial charge layer for each of the two or
more sensing devices into a plurality of spatial dielectric
z-planes along a direction orthogonal to the interface between the
fluid sample and the semiconducting nanostructures, and (2) probing
each of the plurality of z-planes with a specific frequency
selected from a range of frequencies. Specific binding of different
target analytes to the corresponding capture reagents may occur at
known spatial heights within the plurality of interfacial charge
layers for the two or more sensing devices. The sensing circuitry
can be configured to determine the presence and concentration of
each of the different target analytes by measuring the capacitance
and impedance changes at specific frequencies corresponding to
their respective z-planes.
[0206] In some embodiments, the sensing circuitry 160 may be
connected to a computing device 170. The sensing circuitry may or
may not be part of the computing device. The computing device may
be configured to process and/or display results obtained via the
above-described electrochemical detection techniques. For example,
the computing device can be configured to display an
electrochemical signal response 180 which may include a set of
Nyquist plots obtained via the modified EIS technique and/or a set
of Mott-Schottky plots obtained via the Mott-Schottky technique. In
some embodiments, the electrochemical signal response may be
displayed on the computing device 170 for further analysis or data
manipulation by a user.
[0207] In some embodiments, the first target analyte 128-1 may be
cTnI antigen, and the first capture reagent 124-1 may be an
antibody that is specific to the cTnI antigen. The second target
analyte 128-2 may be cTnT antigen, and the second capture reagent
124-2 may be an antibody that is specific to the cTnT antigen. The
semiconducting nanostructures 122-1 and 122-2 on the WEs 120-1 and
120-2 may comprise ZnO nanostructures. The linker reagent 126 may
comprise a DSP linker. The surfaces of the ZnO nanostructures may
be functionalized with the DSP linker for attaching the antibodies
to the nanostructures. Accordingly, the first and second sensing
devices can be used for electrochemical detection of the different
cardiac biomarker Troponin isoforms cTnI and cTnT. Baseline
electrical characterization of the array of sensing devices can be
verified based on an electrochemical impedance response at a
predefined frequency (e.g., 100 Hz). The detection of cTnI and cTnT
in the sample can be achieved using the modified EIS and
Mott-Schottky techniques described as follows.
[0208] B. Modified EIS
[0209] In a conventional EIS technique, impedance changes occurring
at the electrode-electrolyte solution interface can be identified
and quantified. However, the challenge in using conventional EIS
for protein detection has been the inability to distinguish the
impedance signal from background noise as the concentration of the
target protein diminishes in the complex test solutions such as
human serum.
[0210] In the modified EIS technique described in various
embodiments herein, a small AC voltage (for example <100 mV
peak-to-peak) can be applied over a range of frequencies (e.g. from
1 Hz to 15 KHz) across the sensing electrodes (WEs) of a sensing
device or an array of sensing devices. In the presence of a fluid
on the sensing surface, an electrical double layer (EDL) is formed
at the sensing electrode/fluid interface. The capacitive impedance
of the EDL reflects the composition of the
ions/biomolecules/interferents present at the interface. In
conventional EIS, the total capacitive impedance of the EDL is
measured and hence it is not possible to distinguish the signal
from specific binding events and non-specific interactions,
especially when the concentration of the target materials or
analytes is very low as compared to the interferent material.
[0211] In the modified EIS technique disclosed herein, the EDL can
be sectioned along the z-direction, i.e. in the orthogonal
direction to the sensing electrode-electrolyte solution interface
with subnanometer precision. Each spatial z-plane within the
electrical double layer can be probed with a specific frequency.
Since the specific binding of the protein with an immobilized
antibody capture probe is expected to occur at a known spatial
height within the EDL, protein binding even at ultra-low
concentrations can be extracted with precision and accuracy by
measuring the capacitive impedance changes at a specific frequency
corresponding to the z plane in which the protein binding event
occurs. The modified EIS technique disclosed herein is advantageous
in that resolution is not diminished in the presence of complex
media with high concentrations of interferent material.
[0212] In the modified EIS technique, the EDL at the sensing
electrode/electrolyte buffer interface can be fragmented and
analyzed at varying heights from the interface by measuring the
impedance response at multiple frequency planes. Specific
interactions between a target protein and its specific antibody
capture probe can be selectively identified through a maximal
change to the measured impedance at a specific frequency which maps
to the height from the interface where antibody-target analyte
binding happens. The use of the modified EIS technique can enhance
specificity of detection. The use of ZnO can aid in achieving
heightened sensitivity by leveraging the ionic and semiconducting
nature of the semiconducting material. Also, the use of ZnO
nanostructures can enhance signal response as a result of
biomolecule confinement.
[0213] FIG. 8A illustrates fluid sample absorption onto a working
electrode (WE) 120' disposed on a substrate 110. The substrate may
comprise a polyimide membrane. The WE 120' may be a Au electrode
having a Cr/Au surface finish. The WE 120' may be substantially
planar. The WE 120' may be directly functionalized with a linker
126 that selectively immobilizes a capture reagent 124 (e.g., an
antibody) that is specific for a target analyte 128 (e.g., an
antigen). In some embodiments, a blocking reagent 125 may be
optionally included to block excess binding sites on linker 126. A
sample 152 comprising target analytes 128 may be introduced to the
sensing device/array and adsorbed on the WE 120'. FIG. 8B
illustrates z-plane fragmentation using a modified EIS technique on
a plurality of Helmholtz planes at the planar sensor surfaces of
FIG. 8A. Levels L1', L2' and L3' as shown may correspond to
different spatial z-planes which can be probed using logarithmic
frequency scanning (e.g. ranging from 1 Hz-15 kHz).
[0214] FIG. 8C illustrates fluid sample absorption onto a working
electrode (WE) 120 comprising semiconducting ZnO nanostructures 122
disposed on a substrate 110. The WE 120 may functionalized with the
linker 126 that selectively immobilizes a capture reagent 124
(e.g., an antibody) that is specific for a target analyte 128
(e.g., an antigen). In some embodiments, a blocking reagent 125 may
be optionally included to block excess binding sites on linker 126.
A sample 152 comprising target analytes 128 may be introduced to
the sensing device/array and adsorbed on the WE 120. FIG. 8D
illustrates z-plane fragmentation using a modified EIS technique on
a plurality of Helmholtz planes at the EDL interface at the
nanostructured sensor surfaces of FIG. 8C. Levels L1, L2 and L3 as
shown may correspond to different spatial z-planes which can be
probed using logarithmic frequency scanning (e.g. ranging from 1
Hz-15 kHz).
[0215] Comparing FIGS. 8B and 8D, it can be observed that the
height L1 of the semiconducting ZnO nanostructures is greater than
the height L1' of the planar Au electrode layer. Accordingly, the
semiconducting ZnO nanostructures can increase the z-height or
profile of the working electrode which is advantageous. For
example, since the specific binding of a target analyte with an
immobilized capture reagent is expected to occur at a known spatial
height within the EDL, binding events at ultra-low concentrations
can be extracted with precision and accuracy by measuring the
capacitive impedance changes at a specific frequency corresponding
to the z plane in which the protein binding event occurs. By
probing the impedance over a larger L1' plane, the modified EIS
technique can maintain its resolution in the presence of complex
media with a high concentration of interfering material.
[0216] The modified EIS technique can be used to fragment the EDL
along the z direction with subnanometer precision by changing the
frequency of measured response for stepwise changes to the applied
potential within the electrochemical window of the ionic liquid
(IL)/electrolyte. Recognition and detection of specific binding
events for different protein biomarkers (e.g. cTn, NT-pro BNP, and
CRP) in a multiplexed manner can be achieved as a result of
dielectric permittivity modulation along the frequency spectrum due
to the zwitterion stabilization effect of the ionic liquids in the
EDL at the IL/ZnO electrode buffer interface. Bode analysis with
collected impedance spectra can be used to identify the frequency
range at which capacitive behavior is dominant. The identified
frequency range in performing a Nyquist analysis can be used to
quantify the effect of charge transfer for varying concentrations
of a target biomolecule. Thus the ZnO surfaces can enhance
biomolecule detection. The maximum impedance change from different
assay steps can be used to design the calibration dose response
curve to correlate the concentration of bound target biomolecules
and the measured changes in impedance.
[0217] C. Simulation and Design
[0218] FIG. 9A shows a 2D schematic geometric model of the sensing
array of FIG. 4 in COMSOL domain with applied boundary conditions.
COSMOL Multiphysics is a finite element software that can be used
to virtually simulate the real-time behavior of the sensing array
to determine its performance. The simulation results can be used to
optimize the design of the multiplexed sensing array to meet
certain desired characteristics. The use of simulations can also
help to reduce fabrication cost and time.
[0219] The COSMOL model encompasses the multi-electrode geometry
constructed in three dimensional space. Simulations are performed
using an AC/DC module with assumption of no magnetic field effects
to establish that the first and second sensing devices of the array
have the same baseline electrical performance. The geometric
structures of each sensing device comprise three microelectrodes
(WE, CE, and RE) built on polyimide substrate and surrounded by a
rectangle made of PBS. Electrical properties of gold are assigned
to both the counter electrodes (CEs) and the reference electrode
(RE). The working electrodes (WEs) are assigned the semiconducting
properties ofZnO. A constant applied potential of 10 mV is set at
the WE. The boundary condition of both the RE and the CEs is set at
zero potential. Electrical insulation with a von Neumann boundary
condition (n.J=0) is applied to the PBS layer. The transient
electric field is assumed to be confined within the multiplexed
electrodes and the surrounding PBS medium and is governed by the
following continuity equation.
.gradient. J = Q j i . e . .gradient. .sigma. E = - .differential.
.rho. .differential. t ##EQU00002##
where .sigma. is the charge density. Based on Ohm's law, a relation
between the current density, J (vector quantity) and the electric
potential, V (scalar quantity) can be established. The electric
field E, can be obtained from the following constitutive relation
and the gradient of the scalar potential V as shown.
D=.epsilon..sub.o.epsilon..sub.rE
E=-.gradient.V
[0220] In the above equations, D is the displacement current,
.epsilon..sub.O is the permittivity of free space and
.epsilon..sub.r is the relative permittivity of the
material/electrolyte used. The discretization of the system into
finite elements is based on physics-controlled mesh generation.
[0221] FIG. 9B shows the current distribution in the multiplexed
sensing array for simulations performed with the above-described
boundary conditions. The surface plot shows uniform distribution of
current density between the electrodes of the sensing array.
Maximum current density is observed near the surface of WEs which
indicates that the output current response measured using a
modified EIS technique is from the WEs. The direction of the white
arrows corroborates that the electric field lines are directed away
from the positive surface and that the performed simulations are
correct.
[0222] FIG. 9C shows the variation in measured current density with
distance between WE and CE in the sensing array along the vertical
dotted lines depicted in FIG. 9A. FIG. 9D shows the variation in
measured current density with distance between WE and RE in the
sensing array along the horizontal dotted line depicted in FIG. 9A.
The results indicate that both WEs exhibit the same performance
along their surfaces and in each three electrode setup. For points
that are measured farther away from the WE, current density
decreases and with a highest value of 1.7.times.10.sup.15 A/m.sup.2
observed at its surface. The simulation results indicate that both
WEs exhibit the same baseline electrical performance under ideal
conditions, and thus placement of the electrodes in the multiplexed
sensing array has minimal to no variation. Surface modification of
the WEs can perturb the charge distribution at the
electrode/electrolyte interface. These perturbations are based on
realignment of electrons or holes in the electrode surface and ions
in the electrolyte solution. Thus, these charge perturbations can
be leveraged towards designing the sensing devices/array described
herein for multiplexed detection of multiple biomarkers.
[0223] D. Baseline Characterization
[0224] FIG. 10A shows the baseline electrochemical response of a
multiplexed sensing array characterized in the presence of a
supporting electrolyte--PBS at 10 mV peak-to-peak at 100 Hz. The
open circuit (OC) potential at both the first and second sensing
devices is measured to establish that the same electric potential
exists on both sensing devices of the array. This corresponds to
the potential experienced at the working electrode relative to the
reference electrode prior to occurrence of an electrochemical
reaction, and is estimated at 0.02 V, i.e. 25.0.+-.1.8 mV in the
first sensing device and 24.6.+-.1.6 mV in the second sensing
device. Similarly, the short circuit (SC) potential is measured in
presence of PBS and was observed at 0.01 V, i.e. 18.2.+-.0.8 mV in
the first sensing device, and 17.2.+-.0.5 mV in the second sensing
device.
[0225] FIG. 10B shows the impedance response at each step of
immunoassay for both the first and second sensing devices of the
array. Upon functionalization of ZnO surfaces with DSP, the thiol
functional group in DSP binds to the Zn sites in the nanostructures
forming Zn--S bond. The charged working electrodes in the presence
of an ionic buffer medium experience alignment of charges at the
electrode surface forming an electrical double layer (EDL). A
modified Randles equivalent circuit can be used to study the
contribution due to capacitive and resistive elements. The charge
conduction between the working electrode and the ionic buffer
constitute the charge transfer resistance (R.sub.ct), and the
resistance offered by the buffer constitutes the solution
resistance (R.sub.s) in the electrochemical signal response. The
amine reactive 8-carbon spacer molecule in DSP is highly resistive
and hence higher impedance response is obtained. The impedance for
the DSP step is increased from baseline impedance of 2 k.OMEGA. to
1792 k.OMEGA. in the first sensing device, and 2.7 k.OMEGA. to 1701
k.OMEGA. in the second sensing device. The difference in impedance
between the first and second sensing devices can be attributed to
density of functionalization and is within the acceptable
coefficient of variation (CV) for electrical sensing arrays (for
example, CV<10%). In some cases, the CV may be 9%, 8%, 7%, 6%,
5%, or less.
[0226] Prior to functionalization, the working electrodes
comprising ZnO nanostructures can be prepared for antibody
immobilization by performing a 3.times. wash with DMSO followed by
3.times.PBS wash. A decrease in impedance observed with PBS wash
post functionalization may be due to the conducting molecules that
are present in the buffer. For these characterization studies, cTnT
is used to establish consistency in electrical performance between
the first sensing device and the second sensing device during the
immunoassay steps. When antibody (.alpha.-cTnT) is immobilized, the
charges in the outer plane realign and this arrangement is
analogous to that of a parallel plate capacitor that constitute
double layer capacitance (C.sub.dl). The impedance response at the
first sensing device and the second sensing device decreased to 9.1
k.OMEGA. and 8.1 k.OMEGA. respectively due to binding of
.alpha.-cTnT to linker molecule. Post wash step with PBS, the
multiplexed sensing array is treated with a blocking buffer
containing a blocking reagent (e.g. 125) to block any unbound DSP
sites, and the measured impedance is 8.1 k.OMEGA. and 7.5 k.OMEGA.
respectively at the first sensing device and the second sensing
device. The order of testing the first and second sensing devices
did not affect the impedance responses of the multiplexed sensing
array. The sensing array is then washed with PBS to prepare it for
performing antigen dose response studies. The noise in the sensing
array is estimated as a change in output signal response between
pre- and post-buffer wash after a superblock step. The
recommendation for signal noise threshold for any electrical
sensing array is usually 3 times the noise, and noise estimation
for both electrochemical detection techniques is described
elsewhere herein.
[0227] Immunoassays for cTnI detection can be performed at the
first sensing device and that for cTnT detection can be performed
at the second sensing device using the array shown in FIG. 5, for
establishing multiplexed and simultaneous detection of cTnI and
cTnT. The sensing array preparation for detection of these cardiac
biomarkers may comprise of the immunoassay steps described
elsewhere herein. The prepared sensing array is first tested with
neat human serum (HS) which consists of zero concentration of
measured protein biomarker to establish zero dose measurement. This
is used to characterize signal change as a function of antigen
binding to antibody immobilized surfaces. Different concentrations
of cTnI antigen starting with the lowest concentration on
.alpha.-cTnI immobilized ZnO nanostructure surface can be tested at
the first sensing device. Similarly, different doses of cTnT
antigen can be tested on .alpha.-cTnT immobilized ZnO nanostructure
surface at the second sensing device. The change in output signal
response for subsequent doses is calculated from zero dose
measurement to obtain a calibration curve for cTnI and cTnT
detection. The percentage change in measured signal is used to
represent the multiplexed sensing array performance. Detection of
cTnI and cTnT can be achieved using both the modified EIS technique
and Mott-Schottky technique described herein.
[0228] E. Electrochemical Signal Responses
[0229] FIGS. 11A and 11B show Nyquist plots representing the
detection of cTnI and cTnT using the multiplexed sensing array of
FIG. 5. The Nyquist plots can be obtained via the modified EIS
technique described herein. A decrease in capacitive impedance is
observed with increasing concentration of tested protein biomarker
as shown in the Nyquist plots. Analysis of corresponding Bode phase
plots reveals the lag in output signal response (59.degree. for
cTnI detection and 62.degree. for cTnT detection) which
corroborates the maximum contribution to output signal response is
dominated by capacitance at the double layer, C.sub.dl. With
increasing concentrations of tested biomarker binding to antibody
immobilized ZnO surfaces, the charge distribution at EDL is
perturbed resulting in a dominating capacitive impedance observed
at 100 Hz.
[0230] FIGS. 11C and 11D show calibration curves representing the
detection of cTnI and cTnT using the multiplexed sensing array of
FIG. 5. Similarly, the calibration curves can be obtained via the
modified EIS technique described herein. The linear response of
detection for cTnI and cTnT is across the tested concentration
ranges 0.1 pg/mL to 1E5 pg/mL. A dynamic change of 58% for cTnI
detection resulting from impedance range is observed between 4.7
k.OMEGA. and 1.9 kQ. Similarly, the range of impedance observed for
cTnT detection is between 5.8 k.OMEGA. and 2.2. k.OMEGA. resulting
in dynamic range of 61% for cTnT detection. The signal noise
threshold is calculated as three times the change in impedance
response between pre- and post-buffer wash post blocking step in
immunoassay. The observed signal noise threshold for cTnI detection
at the first sensing device is 8.8% and for cTnT detection at the
second sensing device is 7.4%. In some embodiments, the lowest
concentration that can reliably be detected using the multiplexed
sensing array is evaluated to be 1 pg/mL for cTnI detection and 0.1
pg/mL for cTnT detection.
[0231] FIGS. 12A and 12B show Mott-Schottky capacitance (1/C.sup.2)
plotted as a function of applied potential for cTnI and cTnT
detection using the multiplexed sensing array of FIG. 5. The
Mott-Schottky plots are obtained with a voltage sweep of -1 V to
+1V and input signal amplitude of 10 mV peak-to-peak at 1000 Hz. A
smaller change in capacitance (1/C.sup.2) with increasing
concentrations of tested doses of cardiac biomarker is obtained.
FIG. 12A shows linear increase in 1/C.sup.2 at potentials higher
than 0.3 V for cTnI detection which is as expected for an n-type
ZnO. At applied potential higher than 0.7 V, the response 1/C.sup.2
reaches its limiting value and hence 0.7 V is chosen to represent
change in 1/C.sup.2 with cTnI antigen binding. A similar response
is observed for cTnT detection as shown in FIG. 12B. The range of
1/C.sup.2 obtained is between 144.8 and 86.4 (1/F).sup.2 for cTnI
whereas for cTnT detection, 1/C.sup.2 values obtained is in the
lower range from 76.12 to 26.38 (1/F).sup.2 with increasing
concentrations of tested dose. The trend from the Mott-Schottky
plots is consistent with the Nyquist plots obtained via the
modified EIS technique.
[0232] FIGS. 12C and 12D show calibration curves representing the
percentage change in Mott-Schottky capacitance with varying
concentrations of cTNI and cTnT. FIG. 12C shows the calibration
curve for cTnI with 47% dynamic change in output response. The
signal noise threshold is estimated at 11.5% and hence the reliably
detected lowest concentration of cTnI with MS is 1 pg/mL.
Similarly, the calibration curve for cTnT detection is shown in
FIG. 12D. The dynamic change of 67% is obtained with detectable
lowest cTnT concentration at 1 pg/mL. The estimated noise threshold
for cTnT array is 9.2%. It is noted that the slightly higher signal
noise threshold on the Mott-Schottky capacitances relative to that
of the modified EIS plots may be due to the microelectrode layout.
An analysis of the Mott-Schottky plots shows donor densities of
10.sup.22 cm.sup.-3 for the semiconducting ZnO nanostructures.
[0233] The sensing devices and arrays described herein are capable
of detecting a target isoform of protein biomarkers in the presence
of other similar protein biomarkers. In some embodiments described
herein, the non-specificity of .alpha.-cTnT for cTnI isoform and
.alpha.-cTnI for cTnT isoform is tested over the range of
concentrations between 0.1 pg/mL and 1E5 pg/mL. The electrochemical
signal responses in FIGS. 11C, 11D, 12C, and 12D indicate that only
the corresponding target isoform shows a decrease in capacitive
impedance (i.e. increase in percentage change in EIS impedance and
Mott-Schottky capacitance) with increasing dose concentration,
while the signal response due to the non-specific isoform is well
within the established signal noise threshold. The non-specificity
of .alpha.-cTnI and .alpha.-cTnT for alternating isoforms with the
modified EIS is shown in FIGS. 11C and 11D respectively, and with
Mott-Schottky in FIGS. 12C and 12D, respectively. In addition to
target protein biomarkers, a test sample may further comprise a
range of different biomolecules and therefore there exists a
probability for the capture reagents to interact with those
biomolecules and interfere in the detection of the target protein.
This cross-reactivity for .alpha.-cTnI and .alpha.-cTnT is tested
on a multiplexed sensing array with BSA using varying
concentrations diluted in HS in absence of protein biomarkers. BSA
is chosen, as albumin is the main protein in human blood plasma.
The measured EIS response is shown in FIGS. 11C and 11D, and the
measured Mott-Schottky capacitance response is shown in FIGS. 12C
and 12D, respectively. The maximum percentage change in impedance
observed with BSA using the modified EIS is 5.8% and 5.5%
respectively with .alpha.-cTnI and .alpha.-cTnT immobilized ZnO
nanostructured sensing surfaces and is well within the established
signal noise threshold. The maximum percentage change in
capacitance observed with BSA using Mott-Schottky is 10% and 7%
respectively with .alpha.-cTnI and .alpha.-cTnT immobilized ZnO
nanostructured sensing surfaces. Although the Mott-Schottky for BSA
shows relatively high signal response, it is still within the
established signal noise threshold. Thus, the multiplexed sensing
array having ZnO nanostructures demonstrates good specificity and
satisfactory level of cross-reactivity for target cardiac
biomarkers. The above also demonstrates the feasibility of
detection in complex biological medium with both the modified EIS
and Mott-Schottky techniques.
[0234] FIG. 13A shows a calibration curve representing the
detection of NT-proBNP using the multiplexed sensing array of FIG.
5. The calibration curve can be obtained via the modified EIS
technique described herein. The linear response of detection for
NT-proBNP is across the tested concentration ranges 0.1 ng/L to 1E5
ng/L. The range of impedance observed for NT-proBNP detection is
between 30.OMEGA. and 120 k.OMEGA. resulting in dynamic range of
75% for NT-proBNP detection. The signal noise threshold is
calculated as three times the change in impedance response between
pre- and post-buffer wash post blocking step in immunoassay. The
observed signal noise threshold for NT-proBNP detection is at 30%.
In some embodiments, the lowest concentration that can reliably be
detected using the multiplexed sensing array is evaluated to be 1
ng/L for NT-proBNP detection. FIG. 13B shows a strong correlation
between NT-proBNP detection using the exemplary sensing array
described herein and NT-proBNP detection using a conventional
enzyme-linked immunosorbent assay (ELISA). As shown in FIG. 13B,
the R.sup.2 value is 0.98 over a tested range from Ing/L to 1000
ng/L.
[0235] V. Sensing Platforms
[0236] A. Diagnostics Reader Device
[0237] Physicians currently use a combination of imaging and
laboratory analysis for disease diagnosis in a clinical setting.
Samples from patients can be tested for a multitude of biomolecular
markers. This type of analysis, while precise and repeatable,
requires significant processing time and hence not applicable for
POC diagnostics. The development of successful sensing device for
POC disease diagnostics relies on four major attributes: rapid
detection, sensitivity of detection, specificity of detection, and
ease of use. The incorporation of these key features can allow
clinicians to efficiently provide the necessary feedback and care
to their patients regarding diagnosis, prognosis and response to
therapy. However, current handheld POC devices for cardiac
biomarkers often lack the ability to provide diagnostics in
real-time and with high accuracy and consistency at patient bedside
outside the ED and hospital environment such as primary care,
assisted/independent living care, and ambulatory environments.
[0238] The above needs can be addressed using the sensing platform
shown in FIG. 14 in accordance with some embodiments. The sensing
platform may be configured to perform immunoassays as described
elsewhere herein.
[0239] Referring to FIG. 14, a sensing platform 1400 may include a
test strip 1410 and a diagnostic reader device 1420. The test strip
may include a sensing device or sensing array. For example, the
sensing array 400 shown in FIG. 4 may be provided on the test
strip. In some cases, the test strip is composed of a material
comprising a plurality of capillary beds such that, when contacted
with a sample fluid, the sample fluid is transported laterally
across the test strip. The sample fluid may be flowed along a flow
path of the test strip from a proximal end to the distal end of the
test strip. The sample is flowed by capillarity or wicking.
Non-limiting examples of test strips may include porous paper, or a
membrane polymer such as nitrocellulose, polyvinylidene fluoride,
nylon, Fusion 5.TM., or polyethersulfone.
[0240] The test strip 1410 may also include a wicking pad 1412. The
wicking pad may be composed of, e.g., filter paper. Other optional
features may include a cover for supporting and/or protecting the
test strip. The cover may be composed of a sturdy material such as
plastic (e.g., high-impact polystyrene). The cover may, e.g., may
protect from inadvertent splashing of a sample onto the test strip
(e.g., when the device is applied to a urine stream), and to
protect the sensitive areas of the test strip (e.g., the sensing
array). The cover may include various openings or windows along the
test strip. For example, the cover may include a sample application
zone 1414 for applying the fluid sample 152 to the wicking pad
1412.
[0241] The test strip may comprise a zone and/or region for
conducting an immunoassay. The test strip may define a flow path.
The zone and/or region for conducting immunoassays in accordance
with the disclosure may be positioned along a flow path of the test
strip such that a fluid sample may be flowed (e.g., by capillarity)
from the sample application zone 1414 on a proximal end of the
strip to a test zone 150 of the sensing array 400. In some
alternative embodiments, instead of transporting the sample via
capillary flow, the fluid sample 150 may be dispensed (e.g. by
pipetting) directly onto the test zone 150.
[0242] A test strip may comprise sensing array that are
functionalized to detect analytes of interest. Test strips
comprising different types of sensing arrays can be provided. The
sensing arrays may have different sensing electrode materials (e.g.
semiconducting materials), linker chemistries, and capture reagents
for binding with a variety of different target analytes, depending
on the desired sensing/biosensing application and end physiological
state to be predicted.
[0243] The diagnostic reader device 1420 can be configured for use
with the test strip. The reader device can be a hand-held
electronic device. The reader device can be configured to receive
the test strip. For example, the test strip can be inserted into a
receiving port or chamber of the reader device, thereby
establishing electrical connection with the reader device. The
reader device may comprise, for example the multiplexer 150,
sensing circuitry 160, and/or computing device 170 shown in FIG. 5.
The reader device can be configured to perform electro-analytical
diagnostics on the test strip substantially in real-time. The
electro-analytical diagnostics may include collecting and analyzing
the electrochemical signal responses as described elsewhere
herein.
[0244] In the example of FIG. 14, the test strip is shown inserted
into the receiving chamber of the reader device. The reader device
can generate measurement results (e.g., concentration or relative
amounts of analytes present in the sample) from a completed assay
performed on the test strip, as described throughout. The reader
device can display the measurement results on a screen 1422 of the
reader device. In some embodiments, data containing the measurement
results can be transmitted from the reader device to a mobile
device 1440 and/or to a server. The data may be transmitted via one
or more wireless or wired communication channels. The wireless
communication channels may comprise Bluetooth.RTM., WiFi, 3G,
and/or 4G networks.
[0245] In some embodiments, the data containing the measurement
results may be stored in a memory on the reader device when the
reader device is not in operable communication with the mobile
device and/or the server. The data may be transmitted from the
reader device to the mobile device and/or the server when operable
communication between the reader device and the mobile device
and/or the server is re-established.
[0246] A network 1460 can be configured to provide communication
between the various components of the embodiments described herein.
The network may be implemented, in some embodiments, as one or more
networks that connect devices and/or components in the network
layout for allowing communication between them. For example, one or
more diagnostic test devices, mobile devices and/or servers may be
in operable communication with one another over a network. Direct
communications may be provided between two or more of the above
components. The direct communications may occur without requiring
any intermediary device or network. Indirect communications may be
provided between two or more of the above components. The indirect
communications may occur with aid of one or more intermediary
device or network. For instance, indirect communications may
utilize a telecommunications network. Indirect communications may
be performed with aid of one or more router, communication tower,
satellite, or any other intermediary device or network. Examples of
types of communications may include, but are not limited to:
communications via the Internet, Local Area Networks (LANs), Wide
Area Networks (WANs), Bluetooth.RTM., Near Field Communication
(NFC) technologies, networks based on mobile data protocols such as
General Packet Radio Services (GPRS), GSM, Enhanced Data GSM
Environment (EDGE), 3G, 4G, or Long Term Evolution (LTE) protocols,
Infra-Red (IR) communication technologies, and/or Wi-Fi, and may be
wireless, wired, or a combination thereof. In some embodiments, the
network may be implemented using cell and/or pager networks,
satellite, licensed radio, or a combination of licensed and
unlicensed radio. The network may be wireless, wired, or a
combination thereof.
[0247] One or more reader devices, mobile devices and/or servers
may be connected or interconnected to one or more databases 1450.
The databases may be one or more memory devices configured to store
data. Additionally, the databases may also, in some embodiments, be
implemented as a computer system with a storage device. In one
aspect, the databases may be used by components of the network
layout to perform one or more operations consistent with the
disclosed embodiments. In some embodiments, the databases 1450 may
include patient databases.
[0248] In some embodiments, one or more graphical user interfaces
(GUIs) 1422 may be provided on the reader device 1420. Additionally
or optionally, the GUIs may be provided on the mobile device 1440.
The GUIs may be rendered on a display screen. A GUI is a type of
interface that allows users to interact with electronic devices
through graphical icons and visual indicators such as secondary
notation, as opposed to text-based interfaces, typed command labels
or text navigation. The actions in a GUI are usually performed
through direct manipulation of the graphical elements. In addition
to computers, GUIs can be found in hand-held devices such as MP3
players, portable media players, gaming devices and smaller
household, office and industry equipment. The GUIs may be provided
in a software, a software application, a web browser, etc. The GUIs
may be provided through a mobile application. The GUIs may be
rendered through an application (e.g., via an application
programming interface (API) executed on the mobile device). The
GUIs may show images that permit a user to monitor levels of
analytes of interest.
[0249] As depicted in FIG. 14, the sensing platform may further
comprise means for transmitting data generated by the reader device
and sensing array. In some cases, the data may be transmitted to
and/or read from a mobile device (e.g., a cell phone, a tablet), a
computer, a cloud application or any combination thereof. The data
may be transmitted by any means for transmitting data, including,
but not limited to, downloading the data from the system (e.g.,
USB, RS-232 serial, or other industry standard communications
protocol) and wireless transmission (e.g., Bluetooth.RTM., ANT+,
NFC, or other similar industry standard). The information may be
displayed as a report 1430. The report may be displayed on the
screen 1422 of the reader device 1420 or a computer. The report may
be transmitted to a healthcare provider or a caregiver. In some
instances, the data may be downloaded to an electronic health
record. Optionally, the data may comprise or be part of an
electronic health record. For example, the data may be uploaded to
an electronic health record of a user of the devices and methods
described herein. In some cases, the data may be transmitted to a
mobile device and displayed for a user on a mobile application.
[0250] Data collected by and transmitted by the reader device may
include results of the immunoassay test performed on the test
strip. For example, the data may include the concentrations of
different analytes present in a sample. The concentrations may
include relative concentrations or absolute concentrations. For
example, the GUI 1422 in FIG. 14 shows the levels of different
markers such as PCT, CRP, IL-6, and LBP. The data may also include
an outcome such as a diagnostic outcome or a prognostic outcome.
The data may also include alerts to the user (e.g. critical, alert,
safe). In some cases, the alerts may be color-coded to generate
awareness to the user.
[0251] Additional data that may be transmitted by the reader device
include, without limitation, patient information/details, test
settings, device metrics, device setup, time and date of the
immunoassay tests, system status (testing temperature, battery
status, system self-testing and calibration results), error codes
or error messages, etc.
[0252] Current handheld POC devices typically offer detection of a
single biomarker on a single parameter test strip or cartridge. In
contrast, the sensing platform 1400, particularly the sensing array
400 with multiplexer 150 and sensing circuitry 160, can provide
simultaneous detection of multiple biomarkers for rapid diagnostic
and prognostic on a single electrochemical test strip. The
simultaneous and multiplexed detection of multiple biomarkers on a
single electrochemical test strip obviates the need to use multiple
discrete test strips for detecting different biomarkers.
[0253] Additionally, the sensing platform 1400 is capable of
analyzing multiple biomarkers using very small volumes (e.g.
.ltoreq.30 .mu.L) of the fluid sample (e.g. finger-pricked blood)
performed substantially in real-time at the patient's bedside.
[0254] The sensing platform can lower health care costs through
reduced cost of the disposable test strip for multiple biomarker
detection, and providing diagnostic and prognostic analysis at the
patient bedside in non-clinical environments thus generating
savings on physician costs and hospitalization costs. The data
analyzed can be securely transmitted to a secure cloud server for
the primary physician managing the patient to be able to access,
review, and manage guidance and therapies. In the example of FIG.
14, the sensing platform can aid in assessing congestive heart
failure (CHF) risk based on the measured levels of the different
markers, and is therefore of immediate benefit to primary care and
ED physicians. Furthermore, rapid availability of the immunoassay
testing can facilitate a rule-out protocol in a busy emergency
department.
[0255] An example of a POC application using the sensing platform
1400 is next described. A disposable sensing array comprising of
IL/ZnO hybrid liquid/solid semiconducting electrode, is
functionalized with antibodies that are receptors for the panel of
protein biomarkers to be tested. A test sample comprising of
.ltoreq.20 .mu.L (1-2 drops) blood serum, blood plasma can be
dispensed onto the sensor electrodes through standard capillary
wicking methods common to lateral flow immunoassays, which yields
immunoassay formation at the RTIL/ZnO-buffer interface. The sensing
array can be connected to sensing circuitry in the reader device.
The sensing circuitry may include a potentiostat, and the reader
device may be a hand-held electronic device. After an incubation
period sufficient for diffusion limited processes, the sensing
circuitry in the reader device measures the impedance over a range
of frequencies in the electrochemical window of the RTIL. Based on
reference sigmodial calibration, the concentration of a panel of
protein biomolecules (e.g., cTn, NT-proBNP, and CRP) can be
determined and displayed on the reader device. The sensing platform
1400 is capable of ultrasensitive detection of Troponin and
NT-proBNP cardiac markers with high specificity and minimal
cross-reactivity in human serum samples. The protein binding and
detection process for Troponin and NT-proBNP can be achieved by
using a single capture immunoassay (e.g., primary monoclonal
antibody-antigen interaction) without the use of any secondary
antibody.
[0256] In another embodiment, the sensing platform 1400 can be used
in aptasensing for K+ detection. Aptamer oligonucleotides that
contain single or multiple guanine-rich segments are known to form
specific four-stranded helical conformations in solution with an
extraordinary selectivity for potassium. In the absence of
potassium, the aptamer containing multiple guanine-rich segments
adopts a random-coil structure that upon exposure to potassium ion
(K+) solution displaces the equilibrium in favor of the
G-quadruplex form, the G-quadruplex being a conformation of
guanine-rich DNA resulting from the association of sets of four
guanine residues into planar arrays. The sensing platform 1400 is
capable of higher sensitivity and specificity in the detection of
aptamers, as compared to the use of standard ion-selective
electrodes for electrolyte sensing.
[0257] Accordingly, the sensing platform 1400 can be used for
affinity-based impedimetric sensing of troponin (cTnI, cTnT) and
NT-proBNP using specific antibodies and affinity based amperometric
sensing of K+ and other similar ions using specific aptamers from
human blood. As previously described, the human blood can be
transported by capillary action on the test strip to the test zone.
The test strip can be inserted into the reader device to provide
rapid diagnostic and therapeutic response to a physician at the
patient's bedside. The sensing platform 1400 can be used for
near-patient cardiovascular diagnosis and assessment in primary
care, EDs, assisted/independent living care, and ambulatory
environments, towards real-time detection and monitoring levels of
a panel of cardiac biomarkers (cTnI, NT-proBNP) and sodium,
potassium, calcium levels from finger-pricked capillary blood.
[0258] B. Wearable Device
[0259] In some embodiments, the sensing devices and arrays
described herein may be provided on a wearable sensing platform
1500 as shown in FIG. 15. For example, the sensing system 500 shown
in FIG. 5 may be provided on a wearable device 1510. Examples of
wearable devices may include smartwatches, wristbands, glasses,
gloves, headgear (such as hats, helmets, virtual reality headsets,
augmented reality headsets, head-mounted devices (HMD), headbands),
pendants, armbands, leg bands, shoes, vests, motion sensing
devices, etc. The wearable device may be configured to be worn on a
part of a user's body (e.g., a smartwatch or wristband may be worn
on the user's wrist). The wearable device may include one or more
types of sensors. Examples of types of sensors may include heart
rate monitors, external temperature sensors, skin temperature
sensors, capacitive touch sensors, sensors configured to detect a
galvanic skin response (GSR), and the like.
[0260] In some embodiments, the sensing system on the wearable
device can be capable oftransdermally monitoring alcohol content.
For example, the sensing system can be configured to monitor blood
alcohol levels in real time from ambient perspired sweat. A
wearable device (e.g. in the form of a bracelet) can unobtrusively
house the sensing systems described herein for simultaneous
monitoring of Ethanol and paired Ethyl glucuronide (EtG), Ethyl
Sulfate (EtS), Phosphatidylethanol (PEth) levels from ambient
perspired sweat. The wearable device can be capable of transdermal
measurement of blood alcohol content by detecting and quantifying
ethanol paired with simultaneous detection of non-volatile
metabolites EtG, EtS, PEth, etc. from ambient perspired sweat. This
multi-parameter information can be transmitted via wireless data
transmission from the wearable device to portable, hand-held
devices such as a smart phone. EtG and EtS are stable,
non-oxidative metabolites of alcohol and can be detected in body
fluids including sweat. Simultaneous detection of Ethanol and
paired EtG, EtS in perspired sweat using unobtrusive and
comfortable wearable devices can offer the potential to
dramatically improve the ability to accurately assess the responses
to treatments, and build longer term behavioral patterns of the
individual which is of significant value for research and clinical
purposes.
[0261] The wearable sensing platform can provide enhanced ability
for users and health professionals to collect consumption and
exposure assessment data in a variety of scenarios, leading to a
greater understanding of the relationship between personal alcohol
consumption and exposures and to user physiology, psychology, and
disease origins. This can be advantageous in providing assessments
for susceptible and at-risk groups, such as young adults,
recovering addicts, and people with existing chronic diseases. The
wearable sensing platform can be configured to differentiate
results for varying alcohol consumption in varying social settings,
while collecting data from individuals at the point of exposure. In
some cases, wearable sensing platform can also account for
individual mobility/variability as people move though different,
possibly spatially heterogeneous environments (e.g. via GPS
triangulation).
[0262] Enzyme-based ethanol sensing technologies are generally
based on monitoring of NADH in the case of ADH based sensing
devices and O.sub.2 consumption or H.sub.2O.sub.2 production in the
case of alcohol oxidase (AOX) sensing devices. Alcohol
dehydrogenase (ADH; Alcohol:NAD.sup.+ oxidoreductase, EC 1.1.1.1)
catalyzes the reversible oxidation of primary aliphatic and
aromatic alcohols other than methanol. Alcohol oxidase (AOX;
Alcohol:O.sub.2 oxidoreductase, EC 1.1.3.13) catalyzes the
conversion of alcohols into corresponding aldehydes or ketones, but
not the reverse reaction similar to that catalyzed by the ADH
(Scheme 1a). AOX requires flavin-based cofactors, while ADH
requires NAD-based cofactors. The FAD in AOX is avidly associated
with the redox center of the enzyme and is involved in transferring
the hydride ion originated from alcohol substrate to molecular
oxygen leading to the formation of H.sub.2O.sub.2. The oxidation of
alcohols by AOX is irreversible, due to the strong oxidizing
character of O.sub.2. The NAD.sup.+ (or NADP.sup.+) involved in ADH
catalysis is a strong oxidizing agent that accepts the hydride ion
directly from the substrate during the catalysis and generating the
corresponding reduced form, NADH/NADPH.
[0263] In some embodiments, the sensing system on the wearable
device 1510 is configured for catalytic sensing using amperometric
methods, which can be used to detect the presence of alcohol in
perspired human sweat through either of the above described
mechanisms. The ADH or AOX enzyme would be bound to the sensing
electrode surface through the linker chemistry, and NAD.sup.+ or
FAD.sup.+ co factor would be applied to the sensing electrode
surface. The electrochemical reaction being endothermic (negative
.DELTA.G) will primarily proceed in the presence of the catalyst
and under an applied potential. Thus when alcohol is present in the
solution, the reaction with NAD.sup.+ or FAD.sup.+ takes place at
the sensing electrode surface where the catalyst ADH or AOX is
respectively bound and the resulting electrons transfer is measured
and used to quantify in real-time the amount of alcohol present in
the solution.
[0264] In some embodiments, the sensing system on the wearable
device 1510 is configured for EtG detection in pooled human sweat
using affinity based sensing of bound specific antibodies to Au and
ZnO surfaces using the linker chemistry and with the modified EIS
technique described elsewhere herein.
[0265] The sensing system can employ affinity based impedimetric
sensing of EtG and EtS, and PEth using specific antibodies and
catalytic enzymatic based amperometric sensing of alcohol with
affinity bound enzymes on a multi-configurable electrochemical
sensing platform with human sweat sample. This can be used to
monitor personal alcohol consumption and abstinence, and can also
be used to establish behavioral patterns in social settings.
[0266] FIG. 16 is a flowchart showing a method for continuous,
real-time detection of alcohol, EtG, and EtS in accordance with
some embodiments. A wearable device (e.g. an e-bracelet) can be
configured to receive and perform an immunoassay on a test strip. A
test strip containing bodily fluids may be inserted into the
wearable device, and the total alcohol content (TAC), EtG, and EtS
are measured. Next, the measurements are compared against threshold
values. If the TAC is greater than or equal to the threshold
values, a negative alert may be sent to the user and/or to a
caregiver, while the wearable device continues to measure and
record the EtG and EtS levels periodically. Conversely, if the TAC
is less than the threshold values, the history of previously
recorded negative alerts may be analyzed. The current measured EtG
and EtS levels may be compared with previous readouts, to determine
if there is an increasing or decreasing trend/rate. If there is an
increasing trend/rate in the measured EtG and EtS levels, a
negative alert may be sent to the user/caregiver. If there is a
decreasing trend/rate in the measured EtG and EtS levels, the
wearable device may continue to measure and record the EtG and EtS
levels periodically. When the measured EtG and EtS levels falls
below predefined values set by the user/caregiver, the TAC may be
measured to confirm that TAC levels are below the threshold values,
and a positive alert may be subsequently sent to the
user/caregiver. In some embodiments, the method may include various
steps at which the user is notified by the wearable device whether
the test strip needs to be changed. A person of ordinary skill in
the art will recognize many variations, alterations and adaptations
based on the disclosure provided herein. For example, additional
steps may be added as appropriate. Some of the steps may comprise
sub-steps. Some of the steps may be automated (e.g., autonomous
sensing), whereas some of the steps may be manual (e.g., requiring
manual handling, input or responses from a user). The systems and
methods described herein may comprise one or more instructions to
perform at least one or more steps of method 1500.
[0267] V. Multi-Configurable Modular Sensing Device/Array
[0268] Various modifications can be made to the sensing devices or
arrays described elsewhere herein. In some cases, the sensing
devices or arrays can be modular in nature and customized for
different sensing applications. For example, a substrate can be
modified to receive and interchange thereon a plurality of discrete
sensors. The plurality of discrete sensors may comprise different
capture reagents that are configured to selectively bind to
different target analytes in a fluid sample. Providing a
practically unlimited diversity of discrete sensors can result in
better health monitoring and outcomes for users, for a variety of
biological and chemical sensing applications.
[0269] FIGS. 18A-C show an example of a modular sensing device 1800
in accordance with some embodiments. The device 1800 can be
configured to detect one or more targets in a fluid sample. The
device may include a base module 1810. The base module 1810 may be
similar to the substrate (e.g. 110) described elsewhere herein
except the base module comprises a receiving portion 1812. The
receiving portion may include a recess, cavity, or slot. The base
module can be configured to releasably couple to one or more
discrete sensors 1820 via the receiving portion 1812.
The discrete sensor(s) are configured to be mechanically and
electrically coupled to the base module. The discrete sensor(s) can
be used to determine a presence and concentration of one or more
target analytes in a fluid sample based on detected changes to
electron and ion mobility and charge accumulation when the discrete
sensor(s) are coupled to the base module and the fluid sample is
applied to the sensing device.
[0270] The base module 1810 may include a plurality of electrodes.
For example, the base module may include at least one reference
electrode (e.g. 140) and at least one ground electrode (e.g. 130).
In some embodiments, the receiving portion 1812 may be located in a
region between a ground electrode 130 and a reference electrode
140.
[0271] FIG. 18B shows a plurality of discrete sensors 1820-1
through 1820-n that can be interchangeably coupled to the base
module of FIG. 18A. The plurality of discrete sensors can be be
configured to be interchanged and/or mounted onto the base module
using a quick release mechanism and/or without the use of tools.
FIG. 18C shows an example of a first discrete sensor 1820-1 being
coupled to the base module 1810 via the receiving portion 1812.
[0272] Referring to FIG. 18B, each of the discrete sensors 1820 may
comprise a working electrode 120 having a plurality of
semiconducting nanostructures 122 disposed thereon, and a capture
reagent 124 attached to the semiconducting nanostructures. The
discrete sensors may include the same or different types of
semiconducting nanostructures. The discrete sensors may comprise
different capture reagents (124-1 through 124-n) that are
configured to selectively bind to different target analytes in a
fluid sample. The selective binding is configured to effect changes
to the electron and ion mobility and charge accumulation in
different regions of the semiconducting nanostructures and the
fluid sample. The plurality of discrete sensors can be used for
determining the presence and concentration of the different target
analytes in the fluid sample, as described in many embodiments
elsewhere herein.
[0273] In some embodiments, a first discrete sensor may be
releasably coupled to the base module thereby electrically and
mechanically connecting the first discrete sensor to the base
module. Next, a fluid sample suspected to contain a first target
analyte may be applied to the modular sensing device. The first
discrete sensor can be used to determine a presence and
concentration of the first target analyte in the fluid sample based
on detected changes to electron and ion mobility and charge
accumulation specific to the first target analyte. The first
discrete sensor may be detached from the base module after the
presence and concentration of the first target analyte has been
determined.
[0274] Next, a second discrete sensor may be releasably coupled to
the base module thereby electrically and mechanically connecting
the second discrete sensor to the base module. Another fluid sample
suspected to contain a second target analyte may be applied to the
modular sensing device. The second discrete sensor can be used to
determine a presence and concentration of the second target analyte
in the fluid sample based on detected changes to the electron and
ion mobility and charge accumulation specific to the second target
analyte.
[0275] The modular sensing device of FIGS. 18A-C may be modified
into a modular sensing array for example as shown in FIGS. 19A and
19B. A modular sensing array 1900 can be configured for
simultaneous and multiplexed detection of two or more target
analytes in a fluid sample. The array may include a base module
1910 configured to releasably couple to two or more discrete
sensors. In the example of FIGS. 19A-C, the base module may
comprise (1) a first receiving portion 1912-1 configured to couple
to a first discrete sensor 1820-1, and (2) a second receiving
portion 1912-2 configured to couple to a second discrete sensor
1820-2. The discrete sensors 1810-1 and 1810-2 are configured to be
mechanically and electrically coupled to the base module. Each of
the discrete sensors may comprise a working electrode 120 having a
plurality of semiconducting nanostructures 122 disposed thereon,
and a capture reagent 124 attached to the semiconducting
nanostructures. The plurality of discrete sensors comprises
different capture reagents that are configured to selectively bind
to different target analytes in a fluid sample. The selective
binding is configured to effect changes to the electron and ion
mobility and charge accumulation in different regions of the
semiconducting nanostructures and the fluid sample. The discrete
sensors can be used to determine a presence and concentration of at
least two different target analytes in the fluid sample based on
detected changes to electron and ion mobility and charge
accumulation when the discrete sensors are coupled to the base
module and the fluid sample is applied to the sensing array.
[0276] The base module may comprise at least one reference
electrode and at least one counter electrode. For example, the base
module may comprise counter electrodes 140-1 and 140-2, and a
common reference electrode 130. A first sensing device 1800-1 can
be formed by coupling the first discrete sensor 1820-1 to the first
receiving portion 1812-1. The first sensing device 1800-1 may
comprise the first counter electrode 140-1, the working electrode
120-1, and the reference electrode 130. A second sensing device
1800-2 can be formed by coupling the second discrete sensor 1820-2
to the second receiving portion 1812-2. The second sensing device
1800-2 may comprise the second counter electrode 140-2, the working
electrode 120-2, and the reference electrode 130. Accordingly, the
first and second sensing devices 1800-1 and 1800-2 may share a
common reference electrode. The first sensing device 1800-1 can be
configured to determine the presence and concentration of a first
target analyte, and the second sensing device 1800-2 can be
configured to determine the presence and concentration of a second
target analyte, similar to the embodiments described elsewhere
herein.
[0277] In some embodiments, a method of using a modular sensing
array for detecting one or more target analytes in a fluid sample
may include providing a base module configured to releasably couple
to one or more discrete sensors. The method may also include
coupling the one or more discrete sensors to the base module
thereby electrically and mechanically connecting said discrete
sensors to the base module. The method may further include applying
the fluid sample to the modular sensing array, and using the one or
more discrete sensors to determine a presence and concentration of
the one or more target analytes in the fluid sample based on
detected changes to electron and ion mobility and charge
accumulation specific to each of the one or more target
analytes.
[0278] In some embodiments, the above method may include coupling a
first discrete sensor and a second discrete sensor to the base
module thereby electrically and mechanically connecting the first
and second discrete sensors to the base module. A fluid sample
suspected to contain a first target analyte and a second target
analyte may be applied to the modular sensing array. The first
discrete sensor can be to determine a presence and concentration of
the first target analyte in the fluid sample based on detected
changes to electron and ion mobility and charge accumulation
specific to the first target analyte. Similarly, the second
discrete sensor can be used to determine a presence and
concentration of the second target analyte in the fluid sample
based on detected changes to the electron and ion mobility and
charge accumulation specific to the second target analyte.
[0279] VII. Kits
[0280] Further provided herein are kits which may include any
number of immunoassay test devices and/or reader devices of the
disclosure. In one aspect, a kit is provided for determining
qualitatively or quantitatively the presence and concentration of
at least a first analyte and a second analyte in a fluid sample,
the kit comprising: a) a sensing device or array according to one
or more embodiments of the disclosure; and b) instructions for
using the kit.
[0281] In some cases, a kit may provide a sensing device or array
to enable a user to conduct a test on more than one occasion. In
some cases, a kit may include a plurality of test strips each
configured for a single use (i.e., are disposable). A kit may
include a plurality of test devices to enable a user to perform a
test once a day, once every 2 days, once every 3 days, once every 4
days, once every 5 days, once every 6 days, once every week, once
every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5
weeks, once every 6 weeks once every 7 weeks, once every 8 weeks or
more.
[0282] In some cases, kits may include a plurality of immunoassay
test devices, each capable of detecting different analytes. In some
embodiments, kits may include a plurality of discrete sensors for
detecting different analytes. In a particular embodiment, a kit may
include the sensing array disclosed herein, that is capable of
detecting the presence of cTnI and/or cTnT, NT-proBNP, and CRP in a
biological sample such as blood. In another particular embodiment,
a kit may include a sensing array disclosed herein, that is capable
of detecting the presence and concentration of alcohol content,
EtG, and EtS in a biological sample such as sweat.
[0283] In some cases, kits can be provided with instructions. The
instructions can be provided in the kit or they can be accessed
electronically (e.g., on the World Wide Web). The instructions can
provide information on how to use the devices and/or systems of the
present disclosure. The instructions can provide information on how
to perform the methods of the disclosure. In some cases, the kit
can be purchased by a physician or health care provider for
administration at a clinic or hospital. In other cases, the kit can
be purchased by the subject and self-administered (e.g., at home).
In some cases, the kit can be purchased by a laboratory.
[0284] Kits may further comprise a diagnostic reader device or
wearable device of the disclosure. The diagnostic reader device or
wearable device may be configured to be used with the sensing
devices or arrays of the disclosure. The diagnostic reader device
or wearable device may be configured to be in operable
communication with the sensing devices or arrays.
[0285] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
* * * * *